TW201712022A - 5'-substituted nucleoside compounds - Google Patents

Abstract

The present invention relates to novel 5'-substituted nucleoside compounds, pharmaceutical compositions comprising the compounds, and methods of using the compounds to treat cancer, more particularly for the treatment of cancer, in particular glioblastomas, melanoma, sarcomas, gastric cancer, pancreatic cancer, cholangiocarcinoma, bladder cancer, breast cancer, non-small cell lung cancer, leukemias including acute myeloid leukemia, and lymphomas.

Description

5'-substituted nucleoside compound

The present invention relates to novel 5'-substituted nucleoside compounds which inhibit the activity of protein arginine methyltransferase 5 (PRMT5); pharmaceutical compositions comprising the same; and the use of such compounds for the treatment of physiological disorders, In particular, a method for treating cancer.

Protein arginine methyltransferase (PRMT) is a family of enzymes that increase one or two methyl groups to the vascular nitrogen atom of arginine residues on histones and non-histone proteins. The large number of epigenetic modifications caused by PRMT make it possible to modulate a wide variety of cellular functions including, for example, RNA metabolism, transcriptional regulation, signal transduction, embryonic development, and DNA damage repair. Excessive performance of different PRMTs has been frequently associated with many human cancers. Recently, increased evidence suggests that PRMT5, an important member of the PRMT family, is a potential oncoprotein and is involved in tumor formation. PRMT5 is overexpressed in many tumors, making important oncogenes and cancer survival genes a substrate, and regulating alternative splicing and RNA maturation, which are novel mechanisms important for protein expression. In addition, mantle cell lymphoma was shown to be extremely sensitive to PRMT5 inhibition.

Potential inhibitors of PRMT5 have been known in the literature. See, for example, WO2011/079236, WVO2014/100764, WO2014/100716, WO2014/100695, WO2014/100730, WO2014/100734, and WO2014/100719. In addition, certain 5'-substituted nucleosides are known in the literature. See, for example, WO2001/27114 and WO2013/009735.

New cancer treatment is needed. In particular, there is a need for new cancer treatments for glioblastoma, gastric cancer, pancreatic cancer, bladder cancer, lung cancer, leukemia, and lymphoma. There is still a need to provide alternative PRMT5 inhibitors for the treatment of cancer. Preferably such compounds are capable of enabling The characteristics of optimal administration required for maximal inhibition of tumor cell growth are obtained while being acceptable to the patient. Preferably, such compounds will also be bioavailable.

The present invention provides certain novel 5'-substituted nucleoside compounds which are inhibitors of PRMT5 and which have clinical utility as a single agent or in combination with other anticancer agents for the treatment of different types of cancer, and in detail Glioblastoma, melanoma, sarcoma, gastric cancer, pancreatic cancer, cholangiocarcinoma, bladder cancer, breast cancer, non-small cell lung cancer, leukemia including acute myeloid leukemia and lymphoma.

The present invention provides a compound of the formula:

Wherein: R ^1a is hydrogen, C ₁ -C ₂ alkyl, trifluoromethyl, cyano, chloro or fluoro; R ^1b is hydrogen, chloro or fluoro; R ² is hydrogen or C ₁ -C ₃ alkyl; R ³ is hydrogen, methyl or amine; or a pharmaceutically acceptable salt thereof.

The present invention provides a compound of the formula:

Wherein: R ^1a is hydrogen, C ₁ -C ₂ alkyl, C ₃ -C ₆ cycloalkoxy, trifluoromethyl, cyano, chloro or fluoro; R ^1b is hydrogen, chloro or fluoro; R ² is hydrogen Or C ₁ -C ₃ alkyl; and R ³ is hydrogen, methyl or amine; or a pharmaceutically acceptable salt thereof.

The invention further provides a compound of the formula:

Wherein: R ^1a is hydrogen, C ₁ -C ₂ alkyl, trifluoromethyl, cyano, chloro or fluoro; R ^1b is hydrogen, chloro or fluoro; R ² is hydrogen or C ₁ -C ₃ alkyl; R ³ is hydrogen, methyl or amine; or a pharmaceutically acceptable salt thereof.

The invention further provides a compound of the formula:

Wherein: R ^1a is hydrogen, C ₁ -C ₂ alkyl, C ₃ -C ₆ cycloalkoxy, trifluoromethyl, cyano, chloro or fluoro; R ^1b is hydrogen, chloro or fluoro; R ² is hydrogen Or C ₁ -C ₃ alkyl; and R ³ is hydrogen, methyl or amine; or a pharmaceutically acceptable salt thereof.

The invention further provides a compound of the formula: Or a pharmaceutically acceptable salt thereof.

The invention also provides a compound of the formula: It is crystalline and characterized by an X-ray powder diffraction pattern (Cu radiation, λ = 1.54060 Å), the X-ray powder diffraction pattern comprising a peak of 25.1°, and one or more selected from the group consisting of 17.1°, 13.6°, 20.5 Peaks of the group consisting of °, 24.0°, and 14.5° (2θ +/- 0.2°).

The invention further provides a compound of the formula: Or a pharmaceutically acceptable salt thereof.

The invention further provides a compound of the formula: Or a pharmaceutically acceptable salt thereof.

The invention further provides a compound of the formula: Or a pharmaceutically acceptable salt thereof.

The invention further provides a compound of the formula: Or a pharmaceutically acceptable salt thereof.

The invention also provides a pharmaceutical composition comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable excipient, carrier or diluent.

The present invention provides a method for treating cancer in a patient in need of such treatment, which is more specifically referred to as glioblastoma, melanoma, sarcoma, gastric cancer, pancreatic cancer, cholangiocarcinoma, bladder cancer, breast cancer, non-small cells. Lung cancer, leukemia and lymphoma including acute myeloid leukemia, the method comprising administering to the patient an effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof.

The invention also provides a compound of the invention or a pharmaceutically acceptable salt thereof for use in therapy. Further, the present invention provides a compound of the present invention or a pharmaceutically acceptable salt thereof for use in the treatment of cancer, which is more specifically referred to as glioblastoma, melanoma, sarcoma, gastric cancer, pancreatic cancer, cholangiocarcinoma, Bladder cancer, breast cancer, non-small cell lung cancer, leukemia including acute myeloid leukemia and lymphoma. Further, the present invention provides the use of a compound of the present invention or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of cancer, which is more specifically referred to as glioblastoma, melanoma, sarcoma, gastric cancer, pancreas Cancer, cholangiocarcinoma, bladder cancer, breast cancer, non-small cell lung cancer, leukemia including acute myeloid leukemia and lymphoma.

In detail, the compound is Or a pharmaceutically acceptable salt thereof.

In detail, the compound is Or a pharmaceutically acceptable salt thereof.

In detail, the compound is Or a pharmaceutically acceptable salt thereof.

In detail, the compound is Or a pharmaceutically acceptable salt thereof.

As used herein, "treat/treating/treatment" refers to inhibiting, slowing, stopping or reversing the progression or severity of an existing condition or condition.

As used herein, the term "patient" refers to a warm-blooded animal, such as a mammal. A human, especially a person afflicted with a particular disease, condition or condition.

The following paragraphs describe preferred classes of the invention: a) R ^1a is hydrogen, chloro or cyclopropoxy; b) R ^1b is hydrogen or chlorine; c) R ² is hydrogen or methyl; d) R ³ is an amine group ; e) R ^1a is chlorine, R ^1b is hydrogen, and R ² is hydrogen; f) R ^1a is chlorine, R ^1b is hydrogen, and R ² is methyl; g) R ^1a is hydrogen and R ^1b is hydrogen, And R ² is hydrogen; h) R ^1a is cyclopropoxy, R ^1b is chlorine, and R ² is hydrogen; i) R ^1a is chlorine, R ^1b is hydrogen, and R ³ is an amine group; j) R ^1a Is hydrogen, R ^1b is hydrogen, and R ³ is an amine group; k) R ^1a is a cyclopropoxy group, R ^1b is chlorine, and R ³ is an amine group; 1) R ² is hydrogen and R ³ is an amine group; m) R ² is methyl and R ³ is an amine group; n) R ^1a is chlorine, R ^1b is hydrogen, R ² is hydrogen, and R ³ is an amine group; o) R ^1a is hydrogen and R ^1b is hydrogen, R ² is hydrogen and R ³ is an amine group; p) R ^1a is chlorine, R ^1b is hydrogen, R ² is methyl, and R ³ is an amine group; and q) R ^1a is cyclopropoxy, R ^1b Is chlorine, R ² is hydrogen, and R ³ is an amine group.

The skilled reader will understand that the compounds of the invention may form salts. The compounds of the invention contain a basic heteroatom, in particular an amine, and thus react with any of a number of inorganic and organic acids to form a pharmaceutically acceptable acid addition salt. Such pharmaceutically acceptable acid addition salts and the common methods for preparing them are well known in the art. See, for example, P. Stahl et al., HANDBOOK OF PHARMACEUTICAL SALTS: PROPERTIES, SELECTION AND USE, (VCHA/Wiley-VCH, 2008); SMBerge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Sciences , Vol. 66, No. 1. , January 1977.

The compounds of the invention or salts thereof can be prepared by a variety of procedures known in the art, some of which are illustrated in the Schemes, Preparations, and Examples below. The particular synthetic steps for each of the routes can be combined in various ways or combined with steps from the scheme to prepare a compound or salt of the invention. In the following schemes, the products of each step can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, wet milling, and crystallization.

Some intermediates or compounds of the invention may have one or more pairs of palm or stereosymmetric centers. The present invention encompasses all individual stereoisomers, enantiomers and diastereomers, as well as mixtures of enantiomers and diastereomers of such compounds, including racemates. The compounds of the invention containing at least one palm center are preferably present as a single enantiomer or diastereomer. A single enantiomer or diastereomer may be prepared as a starting material for the palmitic reagent (as illustrated in Scheme I below) or by stereoselective or stereospecific synthesis techniques (eg, in Scheme II below) Illustrated) Preparation. Alternatively, a single enantiomer or diastereomer can be separated from the mixture by standard pair chromatography or crystallization techniques.

Additionally, certain intermediates described in the schemes below may contain one or more oxygen or nitrogen protecting groups. The variable protecting group may be the same or different at each occurrence depending on the particular reaction conditions and the particular transformation to be performed. Protection and deprotection conditions are well known to those skilled in the art and are described in the literature (see, for example, "Greene's Protective Groups in Organic Synthesis ", Fourth Edition, Peter GMWuts and Theodora W. Greene, John Wiley and Sons, Inc. 2007).

Some abbreviations are defined as follows: "A375" refers to a human melanoma-derived cell line; "ACN" refers to acetonitrile; "ATCC" refers to the American Type Culture Collection; "BID" refers to Dosing twice daily; "cat.#" means the catalogue number; "CDI" means 1,1'-carbonyldiimidazole;"CDKN1A" means cyclin-dependent kinase inhibitor 1, p21, p21 ^Cip1 or P21 ^Waf1 ; "CPD" means the value from a sample treated with a compound; "[Cp*IrCl ₂ ] ₂ " means a dimer (pentamethylcyclopentadienyl) ruthenium (III) dimer; "CPM" means counting per minute; "CT" means cycle threshold; "DCC" means N, N'-dicyclohexylcarbodiimide;"DCM" means dichloromethane; "DIAD" means Diisopropyl azodicarboxylate; "DIC" means N,N'-diisopropylcarbodiimide;"DIPEA" means N,N-diisopropylethylamine; "DLBCL" means diffuse Severe B-cell lymphoma; "DMEM" refers to Dulbecco's Modified Eagle's (tissue culture) medium; "DMSO" refers to dimethyl sulfoxide "DNA" means deoxyribonucleic acid; "DNase" means deoxyribonuclease; "cDNA" means complementary DNA; "dNTP" means deoxynucleotide triphosphate; "DTT" means disulfide Threitol; "EDCI" means 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; "EDTA" means ethylenediaminetetraacetic acid; "EGTA" Means ethylene glycol-bis(2-aminoethylether) -N,N,N',N' -tetraacetic acid; "EtOAc" means ethyl acetate; "EtOH" means ethanol or ethyl alcohol "FBS" means fetal calf serum; "GAPDH" means glyceraldehyde-3-phosphate dehydrogenase; "GC" means gas chromatography; " ³ H-SAM" means S-[methyl - ³ H]-adenosyl-L-methionine; "HAT" means hypoxanthine-aminopurine-thymidine; "HATU" means hexafluorophosphate [dimethylamino (triazolyl) [4,5-b]pyridin-3-yloxy)methylene]-dimethylammonium; "HBTU" means hexafluorophosphate o-(benzotriazol-1-yl)-N,N,N ',N'-tetramethyl "HOBt" means hydroxybenzotriazole; "HEC" means hydroxyethyl cellulose; "HEPES" means 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; "hr" Refers to the hour; "HOAt" refers to 1-hydroxy-7-azabenzotriazole; "IC ₅₀ " refers to the concentration of the agent that produces the 50% maximum inhibitory response to the agent; "IVTI" refers to the in vivo Target inhibition; "KLH" refers to keyhole snail cyanide; "three second butyl borohydride solution" refers to lithium (t-butyl) borohydride; "MDM4" refers to mouse double microbody 4 (mouse double minute 4); "MeOH" means methanol; "MEP50" means methylosome protein 50; "min" means minute; "MTBE" means methyl third Butyl ether; "OD" means optical density; "od" means outer diameter; "PAGE" means polypropylene guanamine gel electrophoresis; "PBS" means phosphate buffered saline; "PCR" means polymerase Chain reaction; "PEG" means polyethylene glycol; "pNPP" means 4-nitrophenyl phosphate; "PO" means oral or oral administration; "ppm" means one part per million; "PRMT5" Mean egg White arginine methyltransferase 5; "PyBOP" means (benzotriazol-1-yl-oxy-pyrrolidinyl hexafluorophosphate); "psig" means pound-force per square foot gauge pressure "PTFE" means polytetrafluoroethylene; "PyBROP" means bromine-pyrrolidinium hexafluorophosphate; "QD" means once-a-day or once-a-day administration; "qPCR" is a specified amount of polymerase chain "RNA" means ribonucleic acid; "RPMI" means Roswell Park Memorial Institute; "(R,R)-Ts-DENEB ^TM " means N-[(1R,2R) -1,2-diphenyl-2-(2-(4-methylbenzyloxy)ethylamino)-ethyl]-4-toluenesulfonamide (chloro) ruthenium (II); (R,R)-Ts-DEPEN" means (1R,2R)-(-)-N-(4-toluenesulfonyl)-1,2-diphenylethylenediamine; "RT" means inverse Transcriptase; "SAH" means S-adenosyl-homocysteine; "SAM" means S -adenosyl-methionine; "(S)-CBS catalyst" means (S)-Curry - Corey-Bakshi-Shibata catalyst or (S)-1-butyl-3,3-diphenylhexahydropyrrolo[1,2-c][1,3,2] Azo-heterocyclic pentene; "sf9" means pure-derived grass worm ( Spodoptera frugiperda ) insect cells; "SmD1" refers to small nuclear ribonucleoprotein D1; "SPA" refers to scintillation proximity analysis; "SS" refers to stainless steel; "SWFI" refers to sterile water for injection; "TEA "Tris" and "TRIZMA ^® " means 2-amino-2-(hydroxymethyl)-1,3-propanediol or cis (hydroxymethyl)aminomethane; "TBS" Refers to Tris buffered saline; "TEMPO" means 2,2,6,6-tetramethylpiperidine 1-oxy; "THF" means tetrahydrofuran; "TAME" means N-α-p-toluenesulfonyl- L-arginine hydrochloride; "TPCK" means tolsulfonyl phenyl propylamine hydrazinyl chloride; "wt." means weight; "YSI" means bismuth citrate, and "*" means Mathematical operation of multiplication.

In the following schemes, unless otherwise indicated, all substituents are as previously definition. Reagents and starting materials are readily available to those of ordinary skill in the art. Others may be prepared by standard techniques of organic and heterocyclic chemistry or by procedures as described in the preparations and examples followed, including any novel procedures herein.

Scheme 1 depicts the formation of a compound of formula I. "PG" is a protecting group derived from a hydroxyl group. Such protecting groups are well known and understood in the art.

Coupling of 1-ethylindole nuclear furanose protected with tris under pyrrolopidine in 4-step 1 of Scheme 1 with 4-chloro-7H-pyrrolo(2,3-d)pyrimidine provides protection in step 1. product. Trimethyl methanesulfonate trifluoromethanesulfonate combined with acetimilinic acid, N-(trimethyldecyl)-, trimethylsulfonyl ester can be used as an activator to use a solvent such as ACN. -Chloro-7H-pyrrolo(2,3-d)pyrimidine is coupled to a protected nuclear furanose. In a one-pot, 2-step procedure, the hydroxy group is deprotected using an inorganic base (such as sodium methoxide in MeOH) under basic conditions, and then 2,2-dimethoxypropane is used in a solvent such as acetone. Toluenesulfonic acid monohydrate as an activator, the 2,3-hydroxyl substitution of the ribose backbone is selectively protected as a ketal to obtain the product of substep 2 of Step 1 of Scheme 1, followed by the product of Step 2 of Step 2. The 5' alcohol is oxidized to a carboxylic acid using a solution of iodobenzenediacetate and a catalyst such as TEMPO in a solvent such as ACN at a temperature of from 0 ° C to room temperature for about one hour to obtain a process step 2 step 2 The ketal carboxylic acid product. The Weinreb guanamine product of Step 3 of Scheme 1 can be prepared by using N,O-dimethylhydroxylamine hydrochloride in a suitable solvent such as EtOAc, using such as 2,4,6-three Peptide coupling agent for propyl-1,3,5,2,4,6-trioxatriphosphonium-2,4,6-trioxide. An alternative guanamine such as morpholinamide can be prepared in a solvent such as DCM using a carboxylic acid product of Step 1 of Scheme 1 and a suitable nucleophilic amine such as morpholine using a coupling reagent such as CDI. Those skilled in the art will recognize that there are a variety of methods and reagents for reacting a carboxylic acid with an amine to form a guanamine. For example, in the presence or absence of an organic base such as DIPEA or triethylamine in the presence of a coupling reagent, the reaction of the appropriate amine with the carboxylic acid product of Step 2 of Scheme 1 can provide Scheme 1 Step 3 or Scheme 1 The compound of step 4. Coupling reagents include carbodiimides such as DCC, DIC and EDCI; or carbonyl diimidazoles such as CDI. Amidoxime coupling additives such as HOBt and HOAt can also be used to promote the reaction. In addition, non-nucleophilic anions such as HBTU, HATU, PyBOP and PyBrOP can be used. Or bismuth salts to replace more traditional coupling reagents.

The Wein Lofamide product of Step 1 of Scheme 1 or the morpholinium amine product of Step 1 of Scheme 1 can be treated with Grignard reagent to form the product of Step 1 of Scheme 1 and Step 6 of Scheme 1, respectively. Those skilled in the art will recognize that suitable Grignard reagents can react with guanamine. Or the Grignard reagent can be generated in situ by a commercial Grignard reagent (such as isopropylmagnesium chloride-lithium chloride complex) under appropriate bromine or iodine substituted benzene compounds, resulting in step 1 of Scheme 1 and step 1 of Scheme 1. The product of 6. Alternatively, the Grignard reagent can be generated in situ using a suitable bromine or chlorine substituted benzene compound with magnesium swarf and iodine to provide the product of Step 1 of Scheme 1.

The ketone product of Step 1 of Scheme 1 and Step 6 of Scheme 1 can be reacted in a second Grignard reaction to provide the hydroxy product of Step 7 of Scheme 1, wherein R ^{2 is} not hydrogen and R ^{2 is} as previously defined. The product of Step 1 of Scheme 1 or Step 6 of Scheme 1 is treated with the appropriate alkyl Grinna reagent in a solvent such as THF at a temperature of about 0 ° C and quenched with 1 N HCl or aqueous ammonium chloride to give Scheme 1 The product of step 7.

In the substitution of the product of formula I in step 1 of step 1 of step 1 for R ³ =NH ₂ , the displacement of the 4-chloro group of pyrrolopyrimidine to the amine can be accomplished with NH ₃ (such as 7 N NH _{3 in} MeOH). The reaction can be carried out in a sealed vessel and heated at about 100 ° C in the microwave. Alternatively, an aqueous solution of ammonium hydroxide (about 28-30% by weight in water) can be used to displace the chloride in a sealed vessel at about 80-110 ° C for about 8-24 hours.

In the coupling of step 1 of step 1 of step 1 for R ³ =CH ₃ , the 4-chloro of pyrrolopyrimidine can be converted to a methyl group under the following conditions: using a palladium source under conditions of Pd coupling (such as ruthenium (triphenylphosphine) Palladium (0)) and Al(CH ₃ ) ₃ in a solvent such as THF or 1,4-dioxane under an inert atmosphere at a temperature of about 70-80 °C.

In the hydrogenation of R ³ = H in substep 1 of Scheme 1, step 8, the 4-chloro of pyrrolopyrimidine can be removed under hydrogenation conditions. Hydrogenation of such compounds is well known and understood in the art.

In substep 2 of Step 1 of Scheme 1, under acidic conditions, such as TFA in water or HCl in dioxane and MeOH (such as 4 N ) or 4.99 M in 2-propanol and HCl in water, protected The ketal can be deprotected to give a compound of formula I. Deprotection of such compounds is well known and understood in the art.

Process 2

Or in Scheme 2, for R ² = H, the ketone product of Step 5 or Step 6 of Scheme 1 can be reduced in a diastereomeric controlled manner using a non-preferable or palmitic reducing agent to give a diastereoisomer. Body enriched hydroxyl product. For example, ketone reduction using a solution of lithium dibutyl borohydride (L-SELECTRIDE ^® ) in a solvent such as THF at a temperature of about -78 ° C produces the diastereomerization of step 1 of Scheme 2 A hydroxyl product enriched or in the form of a major product. Or in step 2 of Scheme 2, the palmitic reduction catalyst can selectively produce a hydroxyl product in an enriched or predominantly diastereomeric form, wherein the hydroxyl stereo configuration and the lithium dibutylborohydride solution are used. The hydroxy configuration of the hydroxy product produced by the conditions is reversed. Examples of such a pair of palm catalysts which may be used include (S)-CBS-catalysts using a borane-THF complex at a temperature of about -15 ° C in a solvent such as THF; or alternatively, at about room temperature lower side with formic acid / triethylamine complex-based group of ruthenium (II) catalyst complexes, such as (R, R) -Ts-DENEB TM; or alternatively, in a solvent such as a two phase water / DCM systems The ruthenium (III) complex was used in the mixture at about room temperature. Those skilled in the art will recognize that a suitable ruthenium (III) complex can be generated in situ from commercial [Cp*IrCl ₂ ] ₂ and (R,R)-Ts-DEPEN to provide the product of Step 2 of Scheme 2.

In the Examples of the present invention described in the foregoing it is generally found that the use of a chiral reduction catalyst (S) -CBS or (R, R) -DENEB ^TM or iridium (III) complexes from the product of Step 2 of Scheme 2 The major or enriched diastereomer is the diastereomeric configuration of the hydroxyl center formed.

An alternative synthetic method for producing a compound of the invention employs a light delay reaction (Scheme 2, step 3) involving the reaction of a nucleophile such as 4-nitrobenzoic acid with an alcohol to form an ester. Mitsuno-reactions are well known in the art and involve the use of acids such as 4-nitrobenzoic acid, triphenylphosphine and azodicarboxylates such as diethyl azodicarboxylate (DEAD) or azo Diisopropyl diformate (DIAD)) converts the alcohol to an ester in a polar aprotic solvent such as THF at a temperature of from about 0 ° C to room temperature. During the activation and replacement of the galvanic alcohol with a nucleophile, the stereochemistry of the alcohol is reversed. By using the method of the photo-delay reaction, the enriched diastereomer produced by the reduction of the three second butylborohydride solution in step 1 of Scheme 2 can be converted to the same major diastereomer-enriched having a three-dimensional configuration of the product alcohol, as the resulting catalyst composition in Scheme 2, step 2 using (S) -CBS or (R, R) -Ts-DENEB TM or iridium (III) error.

In step 2, step 5 of Scheme 2, for the product of formula I wherein R ³ =NH ₂ , the ester can be subsequently saponified to an alcohol, and the 4-chloro of pyrrolopyrimidine is reacted in a one-pot reaction with step 8 of Scheme 1. The same way is replaced by an amine. In step 2, substep 2 of Scheme 2, as discussed in substep 2 of Scheme 1, step 8, the protected ketal can be protected under acidic conditions to provide a compound of formula I.

In step 4 of Scheme 2, the product of step 2 of Scheme 2 for R ³ =NH ₂ , CH ₃ or H can be carried out as discussed in substep 1 of Scheme 1 Step 8, or alternatively, aqueous ammonium hydroxide (about 28-30% wt% in water) and dioxane can be used to displace chlorides under continuous flow chemistry using a flow rate of about 200 ° C and a flow rate of 10 mL/min (30 minutes residence time) under heating. Or alternatively, at about 200 ° C and a flow rate of 0.251 mL / min (30 minutes residence time). In step 2 of substep 2 of Scheme 2, the protected ketal can be deprotected as discussed in substep 2 of step 1 of Scheme 1, or alternatively, the protected ketal can be protected under the following conditions: with aqueous HCl (about 6 N in water) using a continuous flow chemistry in a mixture of EtOH/MeOH/EtOAc, heating at about 55 ° C and a flow rate of 1.5 mL/min (20 min retention time) or with aqueous HCl (about 4 N) The compound of formula I is obtained in EtOH at about 82 ° C and a flow rate of 0.288 mL/min (10 min residence time).

Those skilled in the art will recognize that the <^1> H NMR spectrum of the exemplified compounds can generally be used to determine the degree and degree of diastereomeric enrichment or purity of the individual diastereomers produced.

The pharmaceutically acceptable salts of the compounds of formula I may be formed by reacting the appropriate free base of formula I with a suitable pharmaceutically acceptable acid under standard conditions in a suitable solvent, as appropriate. . The formation of such salts is well known and understood in the art.

The compounds of the invention were prepared as illustrated in the examples below. The following preparations and examples further illustrate the invention and represent typical syntheses of the compounds of the invention. The reagents and starting materials are readily available or can be readily synthesized by those of ordinary skill in the art. It should be understood that the preparations and examples are set forth by way of illustration and not limitation, and various modifications may be made by those skilled in the art.

The diastereomeric configuration of the compounds of the invention can be determined by standard techniques such as X-ray analysis, ¹ H nmr and correlation with the residence time of the palmitic HPLC.

LC-ES/MS was performed on an AGILENT ^® HP1100 liquid chromatography system. Electrospray mass spectrometry measurements (obtained in positive and/or negative mode) were performed on a mass selective detector quadrupole mass spectrometer interfaced to HP1100 HPLC. LC-MS conditions (low pH): Column: PHENOMENEX ^® GEMINI ^® NX C18 2.1 × 50 mm 3.0 μm; Gradient: 5-100% B in 3 minutes, then 100% B for 0.75 minutes; column temperature: 50 ° C + /-10 ° C; flow rate: 1.2 mL / min; solvent A: deionized water with 0.1% HCOOH; solvent B: ACN with 0.1% formic acid; wavelength: 214 nm. Alternative LC-MS conditions (high pH): Column: XTERRA ^® MS C18 column 2.1 x 50 mm, 3.5 μm; gradient: 5% Solvent A for 0.25 minutes, gradient from 5% to 100% solvent B in 3 minutes, and 100% solvent B for 0.5 minutes, or 10% to 100% solvent B and 100% solvent B for 0.75 minutes in 3 minutes; column temperature: 50 ° C +/- 10 ° C; flow rate: 1.2 mL / min; solvent A : 10 mM NH ₄ HCO ₃ pH ~ 9-10; solvent B: ACN; wavelength: 214 nm.

NMR spectra were performed on a Bruker AVIII HD 400 MHz NMR spectrometer, obtained as CDCl ₃ or (CD ₃ ) ₂ SO solution reported in ppm (chemical shift δ), using residual solvent [CDCl ₃ , 7.26 ppm; (CD ₃ ) ₂ SO, 2.05 ppm] as a reference standard. When reporting peak multimodality, the following abbreviations can be used: s (single peak), t (triplet), q (quadruple), m (multiplet), br-s or bs (wide singlet) , dd (two doublets), dt (two triplets), and td (three doublets). The coupling constant (J) is reported in Hertz (Hz) when reported.

Preparation 1 Benzoic acid [(2R,3R,4R,5R)-3,4-dibenyloxy-5-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-2-yl Methyl ester

ACN (1.8 L) containing 4-chloro-7H-pyrrolo-(2,3-d)pyrimidine (55.3 g, 0.36 mol) was suspended at room temperature and stirred. Ethyl imidate, N-(trimethyldecyl)-, trimethyldecylalkyl ester (110.8 mL, 0.45 mol) was added dropwise and the mixture was stirred at room temperature under N ₂ for 20 min. Trimethyl decyl trifluoromethanesulfonate (1.0 L, 0.54 mol) was added dropwise, followed by the addition of benzoic acid [(2R,3R,4R,5S)-5-ethyloxy-3,4-di). Benzhydryloxy-tetrahydrofuran-2-yl]methyl ester (272.4 g, 0.54 mol). The mixture was heated at 85 ° C (inherent) for 4 hours. The mixture was cooled to 40 ° C (inherent) and additional benzoic acid [(2R,3R,4R,5S)-5-ethyloxy-3,4-dibenyloxy-tetrahydrofuran-2-yl]- The ester (45.40 g, 90.0 mmol) was added portionwise to the mixture. The reaction mixture was heated at 85 ° C (inherent) for 2 hours. The mixture was stirred for 18 hours at room temperature under N _2. Water (500 mL) and EtOAc (500 mL) were added to the mixture. The resulting organic layer was separated. The aqueous layer was extracted with EtOAc (3×250 mL). The organic extracts were combined and washed with saturated aqueous NaHCO ₃ (500 mL) and a saturated aqueous solution (500 mL) sodium chloride. The organic layer was dried over sodium sulfate, filtered, and then evaporated. Purify by EtOAc (EtOAc)EtOAcEtOAc ^{ES / MS m / z (35} Cl / 37 Cl) 598.0 / 600.0 [M + H] +.

Preparation 2 [(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And [3,4-d][1,3]dioxol-6-yl]methanol

Benzoic acid [(2R,3R,4R,5R)-3,4-dibenylmethoxy-5-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-2- yl] methyl acetate (80.5g, 134.61mmol) were suspended in MeOH (805mL), and treated for 3 hours in 0.5 M sodium methoxide (53.8mL, 26.92mmol) in a solution of MeOH at room temperature. Cool to 0 ° C and treat the mixture with DOWEX ^® 50WX2 resin (to pH < 5). Filtration in vacuo and concentration gave a residue. Wet milling in MTBE (100 mL). The mixture was decanted and the residue obtained was dried to give a white solid. The solid was suspended in acetone (1100 mL), and 2,2-dimethoxypropane (41.5 mL, 336.5 mmol) and p-toluenesulfonic acid monohydrate (25.6 g, 134.6 mmol) were added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. Up to ~1/3 of the solvent was removed under reduced pressure, and DCM (250 mL) and water (100 mL) were added. The organic layer was separated and aqueous was extracted with DCM (2×100 mL). The organic extracts were combined, and washed with saturated aqueous NaHCO _3; (100mL) washed (150mL pH ~ 9) and a saturated aqueous sodium chloride solution. The organic layer was dried over sodium sulfate. Filtration and removal of the solvent under reduced pressure gave a crude mixture. The title compound (26.5 g, 60% yield) eluted elute ^{ES / MS m / z (35} Cl / 37 Cl) 326.0 / 328.0 [M + H] +.

Preparation 3 (3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran [3,4-d][1,3]dioxole-6-carboxylic acid

[(3aR,4R,6R,6aR)-4-(4-Chloropyrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]methanol (60.0 g, 184.2 mmol) was dissolved in ACN (250 mL). Water (180 mL) was added and the mixture was cooled to 0 °C. Iodobenzene diacetate (160 g, 496.8 mmol) was added portionwise at 0 ° C, then TEMPO (14 g, 89.6 mmol) was added portionwise. The mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure and EtOAc EtOAc m. Water (100 mL) was added at 0 ° C and the obtained organic layer was separated. The aqueous layer was extracted with EtOAc (3×100 mL). The organic extracts were combined and washed with aq. 10% EtOAc EtOAc. The organic layer was dried with sodium sulfate, filtered and evaporatedEtOAc The crude material was mixed with hexane (400 mL) and the mixture was stirred at room temperature for one hour. The resulting solid was filtered, EtOAcjjjjjjjj ^{ES / MS m / z (35} Cl / 37 Cl) 340.00 / 342.00 [M + H] +.

Preparation 4 (3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-N-methoxy-N,2,2-trimethyl-3a, 4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxole-6-carboxamide

[(3aR,4R,6R,6aR)-4-(4-Chloropyrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]methanol (45.0 g, 138.1 mmol) was dissolved in ACN (175 mL). Water (133 mL) was added and the mixture was cooled to 0 °C. Iodobenzene diacetate (122.3 g, 379.7 mmol) was added portionwise at 0 ° C, then TEMPO (10.5 g, 67.0 mmol) was added portionwise. The mixture was stirred at room temperature for 1 hour. Water (100 mL) and EtOAc (250 mL) were added at EtOAc. The aqueous layer was extracted with EtOAc (3×100 mL). The organic extracts were combined and washed with water (100 mL) EtOAc The organic layer was dried with sodium sulfate, filtered and evaporated This solid was dissolved in EtOAc (400 mL) and EtOAc (EtOAc, EtOAc. The mixture was stirred at room temperature for 5 minutes, and 1.67 M of 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphine-2,4 was added dropwise. , a solution of 6-trioxide in EtOAc (176.3 mL, 294 mmol). The reaction mixture was stirred at room temperature under N ₂ for 3 days. The reaction mixture was cooled to 0.degree. C. and water (150 mL). The resulting organic layer was separated and aqueous layer was evaporated elut The organic extracts were combined and washed with water (200 mL). The organic layer was dried over sodium sulfate, filtered and evaporated Purification by filtration through EtOAc (EtOAc)EtOAc. ^{ES / MS m / z (35} Cl / 37 Cl) 383.0 / 385.0 [M + H] +.

Preparation 5 [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And [3,4-d][1,3]dioxol-6-yl]-(N-morpholinyl)-methanone

(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And [3,4-d][1,3]dioxole-6-carboxylic acid (63.7 g, 166 mmol) was suspended in DCM (320 mL). 1,1 '-Carbonyldiimidazole (37.7 g, 232 mmol) was added portionwise, and the mixture was stirred at room temperature for 45 minutes. Morpholine (21.7 g, 249 mmol) was added dropwise to the mixture and stirred at room temperature for 3 days. The reaction mixture was diluted with DCM and water (150 mL). The resulting organic layer was separated and aqueous layer was extracted with DCM (3×100mL). The organic extracts were combined and washed with water (100 mL) and sat. The organic layer was dried over sodium sulfate, filtered, and then evaporated. Purification by chromatography eluting with EtOAc EtOAc (EtOAc) ^{ES / MS m / z (35} Cl / 37 Cl) 409.00 / 411.00 [M + H] +.

Preparation 6 [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl- 3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-phenyl-ketone

A solution of 3.0 M phenylmagnesium bromide in diethyl ether (1.79 ml, 5.38 mmol) was added dropwise to [(3aR,4R,6S,6aS)-4-(4-chloropyrrole) at 0 °C over 2 min. [2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxole A solution of ene-6-yl]-(N-morpholinyl)-methanone (1.00 g, 2.45 mmol) in THF (15 mL). Stir at 0 °C for 30 minutes and add 1 N aqueous HCl (7.3 mL). After 3 minutes, the aqueous layer was extracted with DCM. The layers were separated and the organic layer was evaporated under reduced pressure. Purification by silica gel chromatography eluting with EtOAc (EtOAc) ^{ES / MS m / z (35} Cl / 37 Cl) 400.0 / 402.0 [M + H] +.

Preparation 7 [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And [3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methanone

(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-N-methoxy-N,2,2-trimethyl-3a , 4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxole-6-carboxamide (30.0 g, 78.4 mmol) was dissolved in THF (300 mL) and cooled To -10 ° C. A 1.0 M solution of 4-chlorophenylmagnesium bromide in diethyl ether (157 mL, 157 mmol) was added dropwise and the mixture was stirred at room temperature for one hour. The reaction mixture was quenched by EtOAc (EtOAc)EtOAc. The resulting organic layer was separated and aqueous brine evaporated The combined organic extracts were washed with water (250 mL Filtration and removal of the organic filtrate under reduced pressure gave a crude mixture. Purified by EtOAc (EtOAc)EtOAcEtOAcEtOAc ^{ES / MS m / z (35} Cl / 37 Cl) 434.0 / 436.0 [M + H] +.

Alternative preparation 7

(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-N-methoxy-N,2,2-trimethyl-3a , 4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxole-6-carboxamide (257.0 g, 617.7 mmol) was dissolved in THF (2570 mL) and cooled To -15 ° C. A solution of 1.0 M 4-chlorophenylmagnesium bromide in 2-methyltetrahydrofuran (1050 mL, 1050 mmol) was added dropwise, and the mixture was stirred at -15 ° C for 1 hour. The reaction mixture was quenched by the addition of saturated aqueous ammonium chloride (1000 mL) and water (500 mL). The resulting organic layer was separated and aqueous brine evaporated The organic extracts were combined and dried over sodium sulfate. Filtration and evaporation of the filtrate under reduced pressure gave a white solid. Purification by recrystallization from EtOAc (EtOAc) ^{ES / MS m / z (35} Cl / 37 Cl) 434.0 / 436.0 [M + H] +.

Preparation 8 [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And [3,4-d][1,3]dioxol-6-yl]-[4-(trifluoromethyl)phenyl]methanone

1-Bromo-4-(trifluoromethyl)benzene (4.0 g, 18 mmol) was added dropwise to a solution of 2.0 M isopropylmagnesium chloride in THF (8 mL, 16 mmol). In THF (10 mL). The mixture was stirred at room temperature for 24 hours. (3aR, 4R, 6S, 6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-N-methoxy-N was added dropwise to the mixture at room temperature. 2,2-Trimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-carboxamide (3.4 g, 8.9 mmol) The solution in THF (14 mL) was stirred at room temperature for 2 h. Cool to 0 ° C and quench the reaction with a 10% EtOAc solution (100 mL). MTBE (200 mL) was added and the mixture was stirred for 20 min. The two phases were separated and the aqueous layer was extracted with MTBE. The combined organic extracts were washed with a saturated aqueous solution of ammonium chloride, water and saturated aqueous sodium chloride. The organic layer was dried with sodium sulfate, filtered, and then evaporated The residue was purified by EtOAc EtOAc EtOAc EtOAc ES/MS m/z ( ³⁵ Cl/ ³⁷ Cl) 468.00/470.00 [M+H] ⁺ .

Preparation 9 [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And [3,4-d][1,3]dioxol-6-yl]-(3-chlorophenyl)methanone

1-Chloro-3-iodobenzene (0.42 g, 1.7 mmol) was dissolved in THF (5 mL) and cooled to EtOAc. A solution of 1.3 M isopropylmagnesium chloride-lithium chloride complex in THF (1.23 mL, 1.6 mmol) was added dropwise over 10 min and stirred at 0 ° C for 1 hour. Add [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4 at 0 °C, 6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(N-morpholinyl)-methanone (0.5 g, 1.2 mmol) in THF Solution in (5 mL). After stirring at 0 ℃ 1 hour by the addition of 1 N aqueous HCl (2mL) mixture is quenched. The resulting mixture was diluted with EtOAc and brine was evaporated. The organic layer was dried with sodium sulfate, filtered, and evaporated. Purification by chromatography eluting with EtOAc EtOAc (EtOAc) ^{ES / MS m / z (35} Cl / 37 Cl) 434.2 / 436.2 [M + H] +.

Preparation 10 [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And [3,4-d][1,3]dioxol-6-yl]-(4-chloro-2-fluoro-phenyl)methanone

4-Chloro-2-fluoro-1-iodo-benzene (1.9 g, 7.5 mmol) was dissolved in THF (50 mL) and cooled to EtOAc. A solution of 1.3 M isopropylmagnesium chloride-lithium chloride complex in THF (6.3 mL, 8.2 mmol) was added and stirred at 0 ° C for 1 hour. Add [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4 at 0 °C, 6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(N-morpholinyl)-methanone (2.8 g, 6.8 mmol) in THF Solution in (10 mL). After stirring at 0 ℃ 5 hours by the addition of 1 N aqueous HCl (9.09mL, 9.09mmol) the mixture was quenched. The resulting mixture was diluted with EtOAc and brine was evaporated. The organic extract was dried over sodium sulfate, filtered, and then filtered. Purification by chromatography eluting with EtOAc (EtOAc)EtOAc ^{ES / MS m / z (35} Cl / 37 Cl) 452.0 / 454.0 / 456 [M + H] +.

Preparation 11 [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And [3,4-d][1,3]dioxol-6-yl]-(3-ethylphenyl)methanone

A solution of 0.5 M 3-ethylphenylmagnesium bromide in THF (5.8 mL, 2.9 mmol) was added dropwise to [(3aR,4R,6S,6aS)-4-(4) at 0 °C over 10 min. -Chloropyrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3] A solution of oxol-6-yl]-(N-morpholinyl)-methanone (1.0 g, 2.5 mmol) in THF (10 mL). Stirred for 30 min at 0 ℃ and by addition of 1 N HCl solution (3.9mL, 3.9mmol) quenched. Dilute with water and extract the aqueous layer with EtOAc. The organic extract was dried over sodium sulfate, filtered, and then evaporated. The residue was purified by EtOAc EtOAc elut elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 428/430 [M + H] +.

Preparation 12 4-[(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a -tetrahydrofuro[3,4-d][1,3]dioxole-6-carbonyl]benzonitrile

4-Bromobenzonitrile (1.36 g, 7.40 mmol) was dissolved in THF (10 mL). A solution of 1.3 M isopropylmagnesium chloride-lithium chloride complex in THF (6.6 mL, 8.53 mmol) was added and stirred at 0 °C for 2 h. This mixture was added to (3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-N-methoxy by cannula at 0 °C. -N,2,2-trimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxole-6-carboxamide (2.18 g, 5.68 mmol) in THF (14 mL). Warm to room temperature and stir for 2 hours. It was quenched by the addition of 1 N aqueous HCl (9.09 mL). The reaction mixture was diluted with EtOAc and water. The layers were separated and the aqueous layer was extracted with EtOAc. The organic extracts were combined and washed with a saturated aqueous solution of sodium bicarbonate and saturated aqueous sodium chloride. The organic layer was dried with MgSO4, filtered and evaporated. The residue was purified by EtOAc EtOAc elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 425.0 / 427.0 [M + H] +.

Preparation 13 [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And [3,4-d][1,3]dioxol-6-yl]-(3,4-difluorophenyl)methanone

Add 4-bromo-1,2-difluoro-benzene (2.21 g, 1.29 mL, 11.4 mmol) to a solution of 2.0 M isopropylmagnesium chloride in THF (6.05 mL, 12.1 mmol) at -15 °C Medium and stirred for 15 minutes. The reaction mixture was warmed and stirred at 0 ° C for 1 hour. This mixture was added to (3aR, 4R, 6S, 6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-N-methoxy-N,2 at 0 °C. ,2-trimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-carboxamide (1.25 g, 3.27 mmol) In a solution of THF (16.3 mL). After 15 minutes, by the addition of 1 N aqueous HCl (5mL) quenched. The reaction mixture was diluted with EtOAc (30 mL) and EtOAc. The layers were separated and aqueous was extracted with EtOAc EtOAc. The organic extracts were combined and washed with a saturated aqueous solution of sodium bicarbonate and saturated aqueous sodium chloride. The organic layer was dried with MgSO4, filtered and evaporated. The residue was purified by EtOAc EtOAcjjjjjjj ^{ES / MS m / z (35} Cl / 37 Cl) 436,438 [M + H] +.

Preparation 14 [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And [3,4-d][1,3]dioxol-6-yl]-(3,4-dichlorobenzene Ketone

Add 1-bromo-3,4-dichlorobenzene (1.04 g, 4.57 mmol) to 2.0 M isopropylmagnesium chloride-lithium chloride complex in THF (1.2 mL, 2.4 mL). In the solution. After 15 minutes, the mixture was warmed to 0 ° C and stirred for 1 hour. This mixture was added to (3aR, 4R, 6S, 6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-N-methoxy-N,2 at 0 °C. ,2-trimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-carboxamide (0.50 g, 1.31 mmol) The solution in THF (6.5 mL) was stirred for 10 min. Quenched with 1 N aq. EtOAc (5 mL)EtOAcEtOAc Stir vigorously for 15 minutes at room temperature. The layers were separated and aqueous was extracted with EtOAc EtOAc. The organic extracts were combined and washed with a saturated aqueous solution of sodium bicarbonate and saturated aqueous sodium chloride. The organic layer was dried over MgSO.sub.4, filtered and evaporated. Purification by chromatography eluting with EtOAc (EtOAc)EtOAc ^{ES / MS m / z (35} Cl / 37 Cl) 468/470 [M + H] +.

Preparation 15 [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And [3,4-d][1,3]dioxol-6-yl]-(p-tolyl)methanone

A solution of 1.0 M p-tolyl bromide in THF (2.4 mL, 2.4 mmol) was added dropwise to [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[ 2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxole A stirred solution of -6-yl]-(N-morpholinyl)-methanone (500 mg, 1.22 mmol) in THF (6.1 mL). After 1 h, it was quenched with EtOAc EtOAc EtOAc. The organic extracts were combined and washed with a saturated aqueous solution of sodium bicarbonate and saturated aqueous sodium chloride. The organic layer was dried with MgSO4, filtered and evaporated. The residue was purified by EtOAc EtOAc elut elut elut elut elut ES/MS m/z ( ³⁵ Cl/ ³⁷ Cl) 414/416 [M+H] ⁺ .

Preparation 16 [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And [3,4-d][1,3]dioxol-6-yl]-(4-fluorophenyl)methanone

To [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(N-morpholinyl)-methanone (2.0 g, 4.90 mmol) in THF (20 mL) A solution of 1.0 M 4-fluorophenylmagnesium bromide in THF (9.8 mL, 9.8 mmol) was added. Stir at 0 ° C for 15 minutes and then at room temperature for 1.5 hours. The mixture was cooled to 0.degree. C. and saturated aqueous ammonium chloride (12 mL). EtOAc (25 mL) was added and stirred vigorously at room temperature for 15 min. The layers were separated and aqueous was extracted with EtOAc EtOAc. The organic extracts were combined and washed with a saturated aqueous solution of sodium bicarbonate and saturated aqueous sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated. Purification by chromatography eluting with EtOAc (EtOAc)EtOAc ^{ES / MS m / z (35} Cl / 37 Cl) 418/420 [M + H] +.

Preparation 17 [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And [3,4-d][1,3]dioxol-6-yl]-(2-fluorophenyl)methanone

1-Bromo-2-fluorobenzene (2.39 g, 7.88 mmol) was dissolved in THF (15 mL). A solution of 1.3 M isopropylmagnesium chloride-lithium chloride complex in THF (9.1 mL, 11.8 mmol) was added and stirred at 0 ° C for 1.5 h. This mixture was added to [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2- by cannula at 0 °C. Dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(N-morpholinyl)-methanone 2.93 g, 7.17 mmol) in THF (30 mL) EtOAc. The mixture was allowed to warm to room temperature and stirred for 2 hours. The reaction was cooled to 0 ℃ and by the addition of 1 N aqueous HCl (11.5mL) and quenched. The reaction mixture was diluted with EtOAc and water. The layers were separated and the aqueous layer was extracted with EtOAc. The organic extracts were combined and washed with saturated aqueous NaHCO ₃ and saturated aqueous sodium chloride. The organic layer was dried with MgSO4, filtered and evaporated. The crude material was purified by chromatography eluting eluting elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 418.0 / 420.0 [M + H] +.

Preparation 18 [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And [3,4-d][1,3]dioxol-6-yl]-[2-(trifluoromethyl)phenyl]methanone

2-Bromobenzotrifluoride (2.97 g, 13.1 mmol) was dissolved in THF (15 mL). A solution of 1.3 M isopropylmagnesium chloride-lithium chloride complex in THF (8.6 mL, 11.2 mmol) was added and stirred at 0 ° C for 1.5 h. The mixture was warmed to room temperature and stirred for 30 minutes. The mixture was again cooled to 0 ° C and the mixture was added to [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidine by cannula at 0 °C. 7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(N -Morphosyl)-methanone (3.05 g, 7.46 mmol) in THF (20 mL). The mixture was allowed to warm to room temperature and stirred for 1 hour. The mixture was cooled to 0 ℃ and was added 1 N aqueous HCl (14.9mL). The mixture was diluted with EtOAc and water. The layers were separated and the aqueous layer was extracted with EtOAc. The organic extracts were combined and washed with saturated aqueous NaHCO ₃ and saturated NaCl. The organic layer was dried with MgSO4, filtered and evaporated. Purification by silica gel chromatography eluting with EtOAc (EtOAc) ^{ES / MS m / z (35} Cl / 37 Cl) 468.0 / 470.0 [M + H] +.

Preparation 19 [(3aR,4R,6S,6aS)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl- 3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-[4-chloro-3-(cyclopropoxy)phenyl Ketone

A mixture of magnesium swarf (4.97 g, 204 mmol) and iodine (920 mg, 3.63 mmol) in THF (150 mL) was stirred for 10 min. A solution of 4-bromo-1-chloro-2-(cyclopropoxy)benzene (46.0 g, 186 mmol) in THF (400 mL) was slowly added dropwise, and the mixture was refluxed for 30 min. Cool to 40 ° C and slowly add this mixture dropwise to (3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-N over 10 minutes. -methoxy-N,2,2-trimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxole-6-carboxamide (35.0 g, 91.6 mmol) in THF (400 mL). The resulting mixture was stirred at room temperature for 30 minutes. Cool to 0 ° C and quench the reaction mixture by adding saturated aqueous ammonium chloride (200 mL) and EtOAc (EtOAc). The resulting organic layer was separated and aqueous layer was extracted with &lt;RTI ID=0.0&gt; The combined organic extracts were washed with water (150 mL Filtration and concentration of the filtrate under reduced pressure gave a crude mixture. Purification by wet milling from EtOAc (EtOAc) (EtOAc) ^{ES / MS m / z (35} Cl / 37 Cl) 490.0 / 492.0 [M + H] +. _{^{1 H NMR (300MHz, CDCl 3}} ) δ 0.85-0.89 (m, 4H), 1.44 (s, 3H), 1.69 (s, 3H), 3.74-3.83 (m, 1H), 5.46-5.48 (m, 2H) , 5.68 (dd, J = 2.2, 6.1 Hz, 1H), 6.39 (d, J = 0.6 Hz, 1H), 6.59 (d, J = 3.6 Hz, 1H), 7.34 - 7.42 (m, 3H), 7.65 ( d, J = 1.8 Hz, 1H), 8.45 (s, 1H).

Preparation 20 (1R)-1-[(3aR,4R,6S,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4 ,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-(4-chlorophenyl)ethanol

[(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methanone (15.9 g, 36.6 mmol) was dissolved in THF (175 mL) Medium and cooled to 0 °C. A solution of 3.4 M methylmagnesium bromide in 2-methyltetrahydrofuran (21.53 mL, 73.3 mmol) was added dropwise and the mixture was stirred at 0 ° C for 2 hr. The reaction mixture was quenched with EtOAc EtOAc (EtOAc) The resulting organic layer was separated and aqueous brine evaporated The combined organic extracts were washed with water (100 mL Filtration and concentration of the filtrate under reduced pressure gave a white, white solid. Purification by chromatography eluting with EtOAc / EtOAc (EtOAc) ES/MS m/z ( ³⁵ Cl/ ³⁷ Cl) 450.00 / 452.00 [M+H] ⁺ .

Preparation 21 1-[(3aR,4R,6S,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a - diastereomers of tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-(3,4-dichlorophenyl)ethanol

[(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3,4-dichlorophenyl)methanone (0.50 g, 1.07 mmol) was dissolved in THF ( In 5.3 mL), and cooled to 0 °C. A solution of 3.0 M methylmagnesium bromide in diethyl ether (0.53 mL, 1.60 mmol) was slowly added and stirred at 0 ° C for one hour. It was quenched by the addition of saturated ammonium chloride (20 mL). Diluted with EtOAc (40 mL) and the layers were separated. The aqueous layer was extracted with additional EtOAc (3×40 mL). The organic extracts were combined and washed with a saturated aqueous solution of sodium bicarbonate and then with saturated aqueous sodium chloride. The organic layer was dried with MgSO4, filtered and evaporated. Purification by silica gel chromatography eluting with 25-30% EtOAc EtOAc (EtOAc) ^{ES / MS m / z (35} Cl / 37 Cl) 484/486 [M + H] +.

Preparation 22 1-[(3aR,4R,6S,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a - diastereomers of tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-(2-fluorophenyl)ethanol

[(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(2-fluorophenyl)methanone (0.685 g, 1.64 mmol) was dissolved in THF (20 mL) And the mixture was cooled to 0 °C. A solution of 3 M methylmagnesium bromide in diethyl ether (1.09 mL, 3.27 mmol) was added and stirred at 0 ° C for 2 h. By addition of 1 N HCl solution (3.44 mL) to quench the reaction. The reaction mixture was diluted with EtOAc and water. The layers were separated and the aqueous layer was extracted with EtOAc. The organic extracts were combined and washed with saturated aqueous NaHCO ₃ and saturated aqueous sodium chloride. The organic layer was dried with MgSO4, filtered and evaporated. The material was purified by EtOAc (EtOAc) elut elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 434.0 / 436.0 [M + H] +.

Preparation 23 1-[(3aR,4R,6S,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a - diastereomers of tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-[2-(trifluoromethyl)phenyl]ethanol

[(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-[2-(trifluoromethyl)phenyl]methanone (0.434 g, 0.928 mmol) was dissolved in THF (12 mL) and the mixture was cooled to 0 °C. A solution of 3.0 M methylmagnesium bromide in diethyl ether (0.62 mL, 1.86 mmol) was added and stirred at 0 °C for 30 min. The reaction was quenched by the addition of 1 N aqueous HCI ( 1.95 mL). The reaction mixture was diluted with EtOAc and water. The layers were separated and the aqueous layer was extracted with EtOAc. The organic extracts were combined and washed with saturated aqueous NaHCO ₃ and saturated aqueous sodium chloride. The organic layer was dried with MgSO4, filtered and evaporated. The residue was purified by EtOAc EtOAc EtOAcEtOAcEtOAc ^{ES / MS m / z (35} Cl / 37 Cl) 484.0 / 486.0 [M + H] +.

Preparation 24 1-[(3aR,4R,6S,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a -tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-[4-(trifluoromethyl)phenyl]ethanol

[(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-[4-(trifluoromethyl)phenyl]methanone (0.425 g, 0.908 mmol) was dissolved in THF (10 mL) and the mixture was cooled to 0 °C. A solution of 3.0 M methylmagnesium bromide in diethyl ether (0.45 mL, 1.35 mmol) was added and stirred at 0 ° C for 1 hour. The reaction was quenched by the addition of 1 N aqueous HCI ( 1.45 mL). The mixture was diluted with EtOAc and water. The layers were separated and the aqueous layer was extracted with EtOAc. The organic extracts were combined and washed with saturated aqueous NaHCO ₃ and saturated aqueous sodium chloride. The organic layer was dried with MgSO4, filtered and evaporated. The resulting residue was purified by EtOAc (EtOAc) elute (0.348 g, 79% yield). ^{ES / MS m / z (35} Cl / 37 Cl) 484.0 / 486.0 [M + H] +.

Preparation 25 (S)-[(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6 , 6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methanol

To [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a, at -78 °C, 4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methanone (6.5 g, 15 mmol) in THF A solution of 1.0 M of a solution of lithium dibutylborohydride in THF (19 mL, 19 mmol) was slowly added to a solution. The mixture was stirred for 4 hours under N ₂ at -78 ℃, and then with saturated aqueous ammonium chloride solution (50mL) and quenched. EtOAc (200 mL) was added and the organic phase was separated. The aqueous phase was extracted with EtOAc (3 x 70 mL). The combined organic extracts were washed with water (100 mL) Filtration and concentration of the filtrate under reduced pressure gave a residue. The residue was purified by EtOAc EtOAc elut elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 436.0 / 438.0 [M + H] +.

Preparation 26 (R)-[(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6 , 6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methanol

[(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methanone (4.77 g, 11.0 mmol) and (R,R)- ^{Ts-DENEB TM (357mg, 0.549mmol} ) was suspended in 5: 2 formic acid - triethylamine complex (45mL) in. The mixture was stirred at room temperature for 18 hours. The mixture was diluted with water (300 mL) and DCM (75 mL). The layers were separated and aqueous layer was extracted with DCM (3. The organic extracts were combined and concentrated under reduced pressure. The residue was purified by EtOAc EtOAc elut elut elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 436.0 / 438.0 / 440.0 [M + H] +.

Alternative preparation 26

The mixture of [Cp*IrCl ₂ ] ₂ (8.41 g, 10.45 mmol) and (R,R)-Ts-DEPEN (7.899, 20.91 mmol) in 1.33/1 water/DCM (3972 mL) was stirred at 50 °C 45 minute. Cool to room temperature and add ammonium formate (509.7 g, 7840 mmol). The two-phase mixture was stirred at room temperature for 5 minutes, and [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2- Dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methanone (227.0 g, 522.7 mmol). The mixture was stirred at room temperature for 1 hour. The resulting organic layer was separated and aqueous layer was extracted with DCM The organic extracts were combined and concentrated under reduced pressure to dry The residue was purified by EtOAc EtOAcqqqqq ^{ES / MS m / z (35} Cl / 37 Cl) 436.0 / 438.0 / 440.0 [M + H] +.

Preparation 27 (R)-[(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6 ,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-[4-chloro-3-(cyclopropoxy)phenyl]methanol

Stir a mixture of [Cp*IrCl ₂ ] ₂ (1.58 g, 1.95 mmol) and (R,R)-Ts-DEPEN (1.43 g; 3.90 mmol) in 1/1 water/DCM (860 mL) at 40 °C 45 minutes. Cool to room temperature and add ammonium formate (100 g, 1462 mmol). The two-phase mixture was stirred at room temperature for 5 minutes, and [(3aR,4R,6S,6aS)-4-(4-aminopyrrolo[2,3-d]pyrimidin-7-yl)-2 was added dropwise. ,2-Dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-[4-chloro-3-( A solution of cyclopropoxy)phenyl]methanone (48.8 g, 97.4 mmol) in DCM (100 mL). The mixture was stirred at room temperature for 1 hour. The resulting organic layer was separated and aqueous layer was extracted with DCM The organic extracts were combined and washed with water (200 mL). The organic extract was dried over sodium sulfate, filtered, and then filtered. The residue was purified by EtOAc EtOAc EtOAc elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 492.0 / 494.0 [M + H] +. _{^{1 H NMR (300MHz, d 6}} -DMSO) δ 0.59-0.71 (m, 4H) 1.30 (s, 3H), 1.51 (s, 3H), 3.61-3.65 (m, 1H), 4.20-4.22 (m, 1H ), 4.68 (t, J = 4.9 Hz, 1H), 5.12 - 5.14 (m, 1H), 5.31-5.34 (m, 1H), 6.09 (d, J = 4.7 Hz, 1H), 6.34 - 6.35 (m, 1H), 6.87-6.80 (m, 2H), 7.11 (s, 1H), 7.29 (d, J = 8.1 Hz, 1H), 8.05 (d, J = 3.7 Hz, 1H), 8.70 (s, 1H).

Preparation 28 4-nitrobenzoic acid [(R)-[(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl 3-a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methyl]ester

Stir (S)-[(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2- under N ₂ at 0 °C Dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methanol (10.3 g , 23.7 mmol), a mixture of 4-nitrobenzoic acid (5.94 g, 35.5 mmol) and triphenylphosphine (9.32 g, 35.5 mmol) in THF (120 mL). DIAD (7.19 g, 35.5 mmol) was added dropwise to the mixture and stirred at room temperature for 6 hours. Additional 4-nitrobenzoic acid (1.98,11.8mmol) at room temperature, triphenylphosphine (3.11g, 11.8mmol), and DIAD (2.40g, 11.8mmol), and the mixture was stirred at room temperature under N ₂ 18 hours. The mixture was cooled to 0.degree. C. and quenched with water (100 mL). EtOAc (100 mL) was added and the obtained organic layer was separated. The aqueous layer was extracted with EtOAc (3×75 mL). The combined organic extracts were washed with water (100 mL Filtration and concentration of the filtrate under reduced pressure gave a crude mixture. Purification by chromatography eluting with EtOAc (EtOAc)EtOAc ES/MS m/z ( ³⁵ Cl/ ³⁷ Cl) 585.00 / 587.00 [M+H] ⁺ .

Preparation 29 [(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And diastereomers of [3,4-d][1,3]dioxol-6-yl]-[4-(trifluoromethyl)phenyl]methanol

To [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4 at -78 °C ,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-[4-(trifluoromethyl)phenyl]methanone (35.0 g, A solution of 1.0 M of a solution of lithium dibutylbromide hydride in THF (93.5 mL, 93.5 mmol) was slowly added to a solution of EtOAc (EtOAc). The mixture was stirred at -78 °C for 30 minutes under N ₂ atmosphere, and then warmed to 0 °C. It was quenched with a saturated aqueous solution of ammonium chloride. EtOAc was added and the organic phase was separated. The aqueous layer was extracted twice with EtOAc. The organic extracts were combined and washed with water and a saturated aqueous solution of sodium chloride. The organic layer was dried with sodium sulfate, filtered and evaporated Purify by silica gel chromatography eluting with EtOAc EtOAc EtOAc ES/MS m/z ( ³⁵ Cl/ ³⁷ Cl) 470.00 / 472.00 [M+H] ⁺ .

Preparation 30 4-nitrobenzoic acid [[(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a, 4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-[4-(trifluoromethyl)phenyl]methyl]ester Diastereomer

[(3aR,4R,6R,6aR)-4-(4-Chloropyrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Non-pair of tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-[4-(trifluoromethyl)phenyl]methanol (21.0 g, 44.7 mmol) enantiomer, 4-nitrobenzoic acid (14.9g, 89.4mmol) and triphenylphosphine (23.45g, 89.4mmol) was dissolved in THF (315mL), and stirred under N ₂ at 0 ℃ in. DIAD (18.1 g, 89.4 mmol) was added dropwise to the mixture and stirred at room temperature for 3 hours. Additional 4-nitrobenzoic acid (7.50g, 44.7mmol) at room temperature, triphenylphosphine (11.8g, 44.7mmol), and DIAD (9.1g, 44.7mmol), and stirred at room temperature under N ₂ The mixture was 2 hours. The mixture was cooled to EtOAc (EtOAc) (EtOAc) The resulting organic layer was separated and EtOAc (3 <EtOAc The organic extracts were combined and washed with water (100 mL) and brine. It was dried over sodium sulfate, filtered, and the filtrate was evaporated. The residue was purified by EtOAc (EtOAc) elut elut The title compound (18.5 g, 60% yield) was obtained. ^{ES / MS m / z (35} Cl / 37 Cl) 619.00 / 621.00 [M + H] +.

Preparation 31 [(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And diastereomers of [3,4-d][1,3]dioxol-6-yl]-(3-chlorophenyl)methanol

To [(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4 at -78 °C ,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3-chlorophenyl)methanone (0.35 g, 0.81 mmol) in THF A solution of 1.0 M of a solution of lithium dibutylborohydride in THF (1.05 mL, 1.05 mmol) was slowly added to the solution in (10.7 mL). Was stirred at -78 deg.] C under N ₂ mixture for 10 minutes, and then was added 1 N aqueous HCl (1.3mL). EtOAc (50 mL) was added EtOAc (EtOAc)EtOAc. The organic extract was dried over sodium sulfate, filtered, and then evaporated. The residue was purified by EtOAc EtOAc elut elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 436.2 / 438.2 [M + H] +.

Preparation 32 4-nitrobenzoic acid [[(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a, Diastereoisomerization of 4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3-chlorophenyl)methyl]ester body

[(3aR,4R,6R,6aR)-4-(4-Chloropyrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl- under N ₂ at 0 ° C Diastereomer of 3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3-chlorophenyl)methanol (0.100 g, 0.229 mmol), 4-nitrobenzoic acid (0.76 g, 0.46 mmol) and triphenylphosphine (0.12 g, 0.46 mmol) were stirred in THF (1 mL). DIAD (0.95 g, 0.46 mmol) was added dropwise to the mixture and stirred at room temperature for 1.5 hours. Additional 4-nitrobenzoic acid (1.98g, 11.8mmol), triphenylphosphine (0.015g, 0.057mmol), and DIAD (0.012g, 0.057mmol), and the mixture was stirred for 18 hours at room temperature under N _2. The mixture was cooled to 0 °C. Water (10 mL), EtOAc (10 mL). The aqueous layer was extracted with EtOAc (2×10 mL). The combined organic extracts were washed with water (100 mL) Filter and concentrate the filtrate under reduced pressure. The residue was purified by EtOAc EtOAc elut elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 585.0 / 587.0 [M + H] +.

Preparation 33 4-[[(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl- Diastereomers of 3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-hydroxy-methyl]benzonitrile

4-[(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6, 6a-tetrahydrofuro[3,4-d][1,3]dioxole-6-carbonyl]benzonitrile (1.33 g, 3.14 mmol), and 1.0 M (S)-1-butyl a solution of -3,3-diphenylhexahydropyrrolo[1,2-c][1,3,2]oxazolane in toluene (0.31 mL, 0.31 mmol) dissolved in THF (50 mL) And the mixture was cooled to -15 °C. A solution of 1.0 M borane-THF complex in THF (2.07 mL, 2.07 mmol) was added. The reaction was allowed to warm to rt and the reaction was stirred 17 h. The reaction mixture was cooled to 0.degree. It was diluted with a saturated aqueous solution of ammonium chloride (40 mL) and EtOAc (EtOAc). The layers were separated and the aqueous layer was extracted with additional EtOAc. The organic extracts were combined, dried over magnesium sulfate, filtered and evaporated. The residue was purified by EtOAc EtOAc elut elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 427.0 / 429.0 [M + H] +.

Preparation 34 [(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And diastereomers of [3,4-d][1,3]dioxol-6-yl]-(3,4-dichlorophenyl)methanol

[(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3,4-dichlorophenyl)methanone (490.0 mg, 1.045 mmol), and 1.0 M (S)-1-butyl-3,3-diphenylhexahydropyrrolo[1,2-c][1,3,2]oxazoborole (0.10 mL, 0.10 mmol) in toluene The solution was dissolved in THF (20.9 mL) and the mixture was cooled to -15 °C. A solution of 1.0 M borane-THF complex in THF (0.69 mL, 0.69 mmol) was added. The reaction was allowed to warm to rt and the reaction was stirred 17 h. Quenched by the addition of MeOH (1 mL). Diluted with saturated aqueous ammonium chloride (40 mL) and EtOAc (40 mL). The layers were separated and aqueous layer was extracted with EtOAc EtOAc. The organic extracts were combined and washed with a saturated aqueous solution of sodium bicarbonate and saturated aqueous sodium chloride. The organic extract was dried over MgSO.sub.4, filtered and evaporated. The residue was purified by EtOAc EtOAc elut elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 470/472 [M + H] +.

Preparation 35 (R)-(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6, 6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-phenyl-methanol

[(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-phenyl-methanone (0.866 g, 2.17 mmol) and (R,R)-Ts-DENEB ^TM (70.4 mg, 10.8 mmol) was suspended in a 5:2 formic acid-triethylamine complex (15 mL) and stirred at room temperature for 4 hours. The reaction mixture was diluted with water (60 mL). The aqueous layer was extracted with DCM. The organic extract was evaporated under reduced pressure. The residue was purified by EtOAc EtOAc elut elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 402.0 / 404.0 [M + H] +.

Preparation 36 [(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And diastereomers of [3,4-d][1,3]dioxol-6-yl]-(4-chloro-2-fluoro-phenyl)methanol

[(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chloro-2-fluoro-phenyl)methanone (0.50 g, 1.0 mmol) and R,R)-Ts-DENEB ⁽ R) (30 mg, 0.05 mmol) was suspended in 5:2 formic acid-triethylamine complex (10 mL) and stirred at room temperature for 1 hour. The reaction mixture was diluted with water (50 mL) and brine was evaporated. The organic extract was dried over sodium sulfate, filtered, and then filtered. The residue was purified by EtOAc EtOAc elut elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 454.0 / 456.0 / 458.0 [M + H] +.

Preparation 37 [(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And diastereomer of [3,4-d][1,3]dioxol-6-yl]-(3-ethylphenyl)methanol

[(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3-ethylphenyl)methanone (0.975 g, 2.28 mmol) and (R,R) -Ts-DENEB(R ⁾ (74 mg, 0.11 mmol) was suspended in a 5:2 formic acid-triethylamine complex (22 mL) and stirred at room temperature for 4 hours. The reaction mixture was diluted with water (250 mL). The aqueous layer was extracted with DCM. The organic extract was dried over sodium sulfate, filtered, and then filtered. The residue was purified by EtOAc EtOAc elut elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 430.2 / 432.2 [M + H] +.

Preparation 38 [(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And diastereomers of [3,4-d][1,3]dioxol-6-yl]-(3,4-difluorophenyl)methanol

The (R, R) -Ts-DENEB TM (93mg, 0.143mmol) was added at room temperature to [(3aR, 4R, 6S, 6aS) -4- (4- chloro-pyrrolo [2,3-d] pyrimidine -7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-( A mixture of 3,4-difluorophenyl)methanone (1.12 g, 2.57 mmol) and 5:2 formic acid-triethylamine complex (10 mL). The reaction vessel was rinsed with N ₂ and the mixture was stirred at room temperature for 1.5 hours. The resulting mixture was diluted with water (75 mL) and DCM (25 mL) and mixed for 5 min. The layers were separated and the aqueous layer was extracted with DCM. The organic extracts were combined and dried over magnesium sulfate. Filter and concentrate the filtrate under reduced pressure. The residue was purified by EtOAc EtOAc EtOAc elut elut elut elut elut ES/MS m/z 438 [M+H] ⁺ .

Preparation 39 [(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And diastereomer of [3,4-d][1,3]dioxol-6-yl]-(p-tolyl)methanol

At room temperature 5: 2 formic acid - triethylamine complex (4.0 mL) was added to (R, R) -Ts-DENEB TM (31.8mg, 0.048mmol) and [(3aR, 4R, 6S, 6aS) - 4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1, 3] A mixture of m-dioxol-6-yl]-(p-tolyl)methanone (405.0 mg, 0.978 mmol) in 1,4-dioxane (4.0 mL). Stir at room temperature for 4 hours. Diluted with water (15 mL) and EtOAc (3x 40 mL). The organic extracts were combined and washed with a saturated aqueous solution of sodium bicarbonate and saturated aqueous sodium chloride. The organic layer was dried with MgSO4, filtered and evaporated. The residue was purified by EtOAc EtOAc elut elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 416/418 [M + H] +.

Preparation 40 [(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran And diastereomers of [3,4-d][1,3]dioxol-6-yl]-(4-fluorophenyl)methanol

Added (R, R) -Ts-DENEB TM (242mg, 0.372mmol) to [(3aR, 4R, 6S, 6aS) -4- (4- chloro-pyrrolo [2,3-d] pyrimidin-7-yl -2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-fluorobenzene a mixture of ketone (1.41 g, 3.24 mmol), 5:2 formic acid-triethylamine complex (25 mL) and 1,4-dioxane (12 mL), and the reaction mixture was stirred at room temperature for 2 hours. . The mixture was diluted with water (80 mL) and DCM (40 mL) and mixed for 5 min. The layers were separated and aqueous layer was extracted with additional DCM (2×40 mL). The organic extracts were combined, dried over sodium sulfate, filtered and evaporated. The residue was purified by EtOAc EtOAcjjjjjjj ES/MS m/z 420 [M+H] ⁺ .

Preparation 41 (R)-[(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4, 6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-phenyl-methanol

Stir (R)-[(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-di in a sealed reaction vessel Methyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-phenyl-methanol (0.399 g, 0.993 mmol) and 7 N NH ₃ in MeOH (15mL, 105mmol) of the mixture. The mixture was microwaved at 100 ° C for 8 hours. The vessel was cooled to room temperature and the solvent was evaporated under a stream of N _2. The residue was purified by EtOAc EtOAc EtOAc elut elut elut elut ES/MS m/z 383.2 [M+H] ⁺ .

Preparation 42 (R)-[(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4, 6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methanol

(R)-4-nitrobenzoic acid [(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl Base-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methyl]ester (10.1 g, 17.3 mmol), ammonium hydroxide (28% in water, 50 mL) and 1,4-dioxane (50 mL) were added to a sealed vessel and heated at 110 ° C for 18 hours. The reaction was cooled to 0.degree. C. and water (50 mL)EtOAc The resulting organic layer was separated and aqueous brine evaporated The organic extracts were combined and dried over sodium sulfate. Filtration and concentration of the filtrate under reduced pressure gave a residue. The residue was purified by filtration through a pad of hexane: EtOAc (1:1). The title compound (6.78 g, 75% yield) elute

Alternative preparation 42

A 300 mL seamless stainless steel tubular reactor (od = 1/8") was placed inside the GC oven and rinsed with 10:10 ammonium hydroxide (28% in water) / dioxane at 10 mL/min over 40 minutes. the back pressure N (800psig) ₂ is applied to the outlet of the reaction system, and the temperature of the GC oven set at 200 ℃. 1L Teledyne ISCO ^TM high pressure injection pump at 10mL / min (30 minutes retention time) pumping (R)-[(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6 , 6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methanol (200.0 g, 458.41 mmol) at a ratio of 3:10 A solution of ammonium hydroxide (28% in water) / dioxane (1300 mL) was passed through the reactor. After depleting the feed solution, 3:10 ammonium hydroxide was pumped at 10 mL/min (28% The water was washed with dioxane (600 mL). The solvent of the collected solution was taken to 1/3 volume under vacuum, water (300 mL) was added and extracted with EtOAc (3×150 mL). Washed with a saturated aqueous solution of sodium chloride, dried over sodium sulfate, filtered, and evaporated. The residue, to give the title compound (189.0g, 95% purity) .ES / MS m / z ( 35 Cl / 37 Cl) 416.0 / 418.0 [M + H] +.

Preparation 43 (1R)-1-[(3aR,4R,6S,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a, 4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-(4-chlorophenyl)ethanol

In a sealed container, (1R)-1-[(3aR,4R,6S,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-di Methyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-(4-chlorophenyl)ethanol (11.4 g, 25.3 mmol) was dissolved in ammonium hydroxide (28% in water, 60 mL) and 1,4-dioxane (60 mL) and warmed at 110 ° C for 18 hours. Cool to room temperature and remove the solvent under reduced pressure. Water (50 mL) and EtOAc (100 mL) were added. The resulting organic layer was separated and aqueous brine evaporated The combined organic extracts were dried with EtOAc EtOAcjjjjjjjjjj ^{ES / MS m / z (35} Cl / 37 Cl) 431.00 / 433.20 [M + H] +.

Preparation 44 [(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Diastereomers of tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-[4-(trifluoromethyl)phenyl]methanol

4-nitrobenzoic acid [[(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2- in a sealed tube Dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-[4-(trifluoromethyl)phenyl The diastereomer of the methyl ester (21 g, 33.9 mmol) was dissolved in ammonium hydroxide (28 wt% in water, 100 mL) and 1,4-dioxane (100 mL) and heated at 110 °C The mixture was 18 hours. The tube was cooled to room temperature, the precipitated solid was collected by filtration, and the cake was washed with water. The filter cake was dissolved in EtOAc (300 mL). The resulting organic layer was washed with water and dried over sodium sulfate. Filtration and concentration of the filtrate under reduced pressure gave a residue. The residue was purified by EtOAc EtOAc (EtOAc) elute 80% yield). ES/MS m/z 451.2/452.2 [M+H] ⁺ .

Preparation 45 [(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Diastereomer of tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3-chlorophenyl)methanol

Stir 4-nitrobenzoic acid [[(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2 in a sealed reaction vessel, 2-Dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3-chlorophenyl)methyl The ester diastereomer (0.105 g, 0.18 mmol), 1,4-dioxane (4 mL) and ammonium hydroxide (30 wt% in water, 6 mL). The reaction was heated to 85 °C. After 24 hours at 85 ° C, it was cooled to room temperature, additional ammonium hydroxide (30 wt% in water, 6 mL) was added and stirred at 95 ° C for an additional 24 hours. Cool to room temperature and add water (50 mL). Extract with EtOAc. The organic extracts were combined, dried over sodium sulfate, filtered and evaporated. The residue was purified by EtOAc EtOAc elut elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 417.2 / 419.2 [M + H] +.

Preparation 46 [(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Diastereomer of tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chloro-2-fluoro-phenyl)methanol

Stir [(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a in a sealed reaction vessel , 4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chloro-2-fluoro-phenyl)methanol A mixture of the phantom (0.12 mg, 0.26 mmol), 1,4-dioxane (2 mL) and ammonium hydroxide (30 wt% in water, 4 mL). The mixture was heated to 80 °C. After 12 hours, the tube was cooled to room temperature and additional ammonium hydroxide (30 wt% in water, 4 mL) was added. The sealed container was further heated at 80 ° C for 3 hours. The reaction was cooled to room rt, diluted with H~~~~ The organic extracts were combined, dried over sodium sulfate, filtered and evaporated. The residue was purified by EtOAc EtOAc elut elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 435.2 / 437.2 [M + H] +.

Preparation 47 [(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Diastereomer of tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3-ethylphenyl)methanol

Stir [(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a in a sealed reaction vessel , diastereomer of 4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3-ethylphenyl)methanol A mixture of (0.370 g, 0.861 mmol), 1,4-dioxane (4 mL) and ammonium hydroxide (30 wt% in water, 6 mL). The mixture was heated to 80 ° C for 12 hours. The vessel was cooled to room temperature and the mixture was diluted with EtOAc EtOAc. The organic extracts were combined, dried over sodium sulfate, filtered and evaporated. The residue was purified by EtOAc EtOAc elut elut elut elut elut elut ES/MS m/z 411.2 [M+H] ⁺ .

Preparation 48 4-[[(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6 , diastereomer of 6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-hydroxy-methyl]benzonitrile

In a sealed reaction vessel, 4-[[(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl Diastereoisomerism of keto-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-hydroxy-methyl]benzonitrile The construct (0.765 g, 1.79 mmol) was dissolved in 1,4-dioxane (4 mL) and ammonium hydroxide (28 wt% in water, 4 mL). The reaction was heated to 85 ° C and stirred for 18 hours. The reaction was cooled to room temperature and concentrated under reduced pressure. Via a silicone gel with 60-75% of 10% 7 N NH in MeOH in the ₃ / MTBE of 9: 1 mixture of / hexane gradient eluting, the resulting residue was purified to give the title compound (0.64g, 88% yield rate). ES/MS m/z 408 [M+H] ⁺ .

Preparation 49 [(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Diastereomer of tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3,4-difluorophenyl)methanol

In the reaction vessel, [(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4 , 6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3,4-difluorophenyl)methanol diastereomer (795.9 mg, 1.76 mmol) was dissolved in 1,4-dioxane (4 mL), and ammonium hydroxide (28 wt% in water, 6 mL) was added. The vessel was sealed and the mixture was heated to 85 ° C with stirring for 18 hours. The vessel was cooled to room temperature and concentrated under reduced pressure. The resulting solid was dissolved in a solution of 2% MeOH in DCM and filtered. The filtrate was concentrated to give the title compound (jjjjd ES/MS m/z 419 [M+H] ⁺ .

Preparation 50 [(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Diastereomers of tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3,4-dichlorophenyl)methanol

In a sealed reaction vessel, [(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a , diastereoisomerism of 4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3,4-dichlorophenyl)methanol The construct (245.0 mg, 0.520 mmol) was dissolved in ammonium hydroxide (28 wt% in water, 2.0 mL) and 1,4-dioxane (2.0 mL). The vessel was heated to 85 ° C and stirred for 18 hours. The mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by EtOAc EtOAc EtOAc elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 451/453 [M + H] +.

Preparation 51 [(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Diastereomer of tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(p-tolyl)methanol

In a sealed reaction vessel, [(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a , 4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(p-tolyl)methanol diastereomer (144 mg, 0.346 mmol) was dissolved in ammonium hydroxide (28 wt% in water, 4.0 mL) and 1,4-dioxane (4.0 mL). The reaction was heated to 85 ° C and stirred for 18 hours. The reaction was cooled to room temperature and concentrated under reduced pressure. The residue was purified by EtOAc EtOAc elut elut elut elut elut elut ES/MS m/z 397 [M+H] ⁺ .

Preparation 52 [(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Diastereomer of tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-fluorophenyl)methanol

Feeding [(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl- to each of the two sealed vessels Diastereomer of 3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-fluorophenyl)methanol (505 mg, 1.20 mmol), ammonium hydroxide (28 wt% in water, 15 mL) and 1,4-dioxane (15 mL). The mixture was heated to 85 ° C and stirred for 18 hours. The mixture was cooled to room temperature and ammonium hydroxide (28 wt% in water, 8 mL) was added. Both mixtures were heated to 75 ° C and stirred for 60 hours. The contents of the two containers were combined and concentrated to dryness under reduced pressure. The resulting residue was dissolved in aq. EtOAc (EtOAc)EtOAc. The filtrate was concentrated to give a residue. The residue was purified by EtOAc EtOAc elut elut elut elut elut ES/MS m/z 401 [M+H] ⁺ .

Preparation 53 1-[(3aR,4R,6S,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6, Diastereomer of 6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-(3,4-dichlorophenyl)ethanol

1-[(3aR,4R,6S,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6, a diastereomer of 6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-(3,4-dichlorophenyl)ethanol ( 150.0 mg, 0.309 mmol), ammonium hydroxide (28 wt% in water, 3.0 mL) and 1,4-dioxane (3.1 mL) were dissolved in a sealed reaction vessel. The reaction was heated to 85 ° C and stirred for 18 hours. The reaction was cooled to room temperature and concentrated under reduced pressure. The residue was purified by EtOAc EtOAc EtOAc elut elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 465/467 [M + H] +.

Preparation 54 1-[(3aR,4R,6S,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6, Diastereomer of 6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-(2-fluorophenyl)ethanol

In a sealed reaction vessel, 1-[(3aR,4R,6S,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl Diastereomerization of -3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-(2-fluorophenyl)ethanol The isomer (0.48 g, 1.1 mmol) was dissolved in 1,4-dioxane (6 mL) and ammonium hydroxide (28 wt% in water, 4 mL). The vessel was heated to 85 ° C and stirred for 22 hours. The mixture was cooled to room temperature and concentrated under reduced pressure. Via a silicone gel with 25-75% of 10% 7 N NH in MeOH in the ₃ / MTBE of 9: 1 mixture of / hexane gradient eluting, the resulting residue was purified to give the title compound (0.35g, 75% yield rate). ES/MS m/z 415.0 [M+H] ⁺ .

Preparation 55 1-[(3aR,4R,6S,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6, Diastereomerization of 6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-[2-(trifluoromethyl)phenyl]ethanol body

In a sealed reaction vessel, 1-[(3aR,4R,6S,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl -3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-[2-(trifluoromethyl)phenyl] The diastereomer of ethanol (0.18 g, 0.37 mmol) was dissolved in 1,4-dioxane (3 mL) and ammonium hydroxide (28 wt% in water, 1 mL). The vessel was heated to 85 ° C and stirred for 22 hours. The mixture was cooled to room temperature and concentrated under reduced pressure. Via a silicone gel with 50-75% of 10% 7 N NH in MeOH in the ₃ / MTBE of 9: 1 mixture of / hexane gradient eluting, the resulting residue was purified to give the title compound (0.14g, 84% yield rate). ES/MS m/z 465.0 [M+H] ⁺ .

Preparation 56 1-[(3aR,4R,6S,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6, Diastereomerization of 6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-[4-(trifluoromethyl)phenyl]ethanol body

In a sealed reaction vessel, 1-[(3aR,4R,6S,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl -3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-[4-(trifluoromethyl)phenyl] Ethanol (0.33 g, 0.68 mmol) was dissolved in 1,4-dioxane (2 mL) and ammonium hydroxide (28 wt% in water, 3 mL). The vessel was heated to 85 ° C and the contents were stirred for 22 hours. The reaction was cooled to room temperature and concentrated under reduced pressure. Via a silicone gel with 25-75% of 10% 7 N NH in MeOH in the ₃ / MTBE of 9: 1 mixture of / hexane gradient eluting, the resulting residue was purified to give the title compound (0.152g, 48% yield rate). ES/MS m/z 465.0 [M+H] ⁺ .

Preparation 57 (R)-[(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4, 6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-[4-chloro-3-(cyclopropoxy)phenyl]methanol

A 59 mL seamless stainless steel tubular reactor (od = 1/8") was placed inside the GC oven with a 1:6 ratio of ammonium hydroxide (28% in water) / dioxane at 2.0 mL/min over 40 minutes. Rinse. Apply N ₂ back pressure (1400-1500 psig) to the outlet of the reaction system and set the temperature of the GC oven to 200 ° C. Use a high pressure 1 L Teledyne ISCO ^TM syringe pump at 0.25 mL/min (30 min. Retention time) pumping (R)-[(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl- 3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-[4-chloro-3-(cyclopropoxy)phenyl A solution of the diastereomer of methanol (35.3 g, 70.2 mmol) in a mixture of 1/6 ammonium hydroxide (28% in water) / dioxane (350 mL) was passed through the reactor. After the solution, the reactor was rinsed with 1:6 ammonium hydroxide (28% in water): dioxane (89 mL), which was pumped at 2.0 mL/min. Water (1400 mL) and EtOAc (500 mL) were added. The aqueous layer was extracted with EtOAc EtOAc EtOAc. S m / z ( ³⁵ Cl / ³⁷ Cl) 473.1/475.1 [M + H] ^{+ 1} H NMR (300 MHz, d ₆ -DMSO) δ 0.73-0.63 (m, 4H), 1.30 (s, 3H), 1.49 (s, 3H), 4.10 (dd, J = 1.6, 6.6 Hz, 1H), 4.66 (dd, J = 4.3, 6.4 Hz, 1H), 5.11 (dd, J = 1.8, 6.2 Hz, 1H), 5.33 ( Dd, J = 3.3, 6.3 Hz, 1H), 6.12 (d, J = 3.3 Hz, 1H), 6.30 (d, J = 4.1 Hz, 1H), 6.64 (d, J = 3.6 Hz, 1H), 6.86- 6.90 (m, 1H), 6.93 (d, J = 1.6 Hz, 1H), 7.14 (s, 2H), 7.28 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 3.6 Hz, 1H), 8.09 (s, 1H).

Preparation 58 (1R)-1-[(3aR,4R,6S,6aR)-2,2-dimethyl-4-(4-methylpyrrolo[2,3-d]pyrimidin-7-yl)-3a, 4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-(4-chlorophenyl)ethanol

To a high pressure reaction vessel under a N ₂ atmosphere, a solution of 2.0 M Al(CH ₃ ) ₃ in toluene (120 μL, 0.24 mmol) was added to (1R)-1-[(3aR,4R,6S,6aR)- 4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1, 3] m-dioxol-6-yl]-1-(4-chlorophenyl)ethanol (0.30 mg, 0.666 mmol), hydrazine (triphenylphosphine) palladium (0) (0.154 g, 0.016 mmol) And a stirred mixture of anhydrous THF (8 mL). The reaction vessel was sealed and the mixture was heated to 70 °C. After 70 deg.] C for 6 hours, the vessel was cooled to room temperature and carefully add 1 N aqueous HCl (15mL). The mixture was diluted with water (15 mL) andEtOAcEtOAc The combined organic extracts were washed with EtOAc EtOAc m. The residue was purified by EtOAc EtOAc EtOAcEtOAcEtOAc ^{ES / MS m / z (35} Cl / 37 Cl) 430.0 / 432.0 [M + H] +.

Preparation 59 [(3aR,4R,6R,6aR)-2,2-dimethyl-4-(4-methylpyrrolo[2,3-d]pyrimidin-7-yl)-3a,4,6,6a- Diastereomer of tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chloro-2-fluoro-phenyl)methanol

A solution of 2.0 M Al(CH ₃ ) ₃ in toluene (120 μL, 2.4 mmol) was added to [(3aR,4R,6R,6aR)-4-(4-chloro) in a high pressure reaction vessel under N ₂ atmosphere. Pyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxan Diastereomer of cyclopentene-6-yl]-(4-chloro-2-fluoro-phenyl)methanol (0.271 mg, 0.597 mmol), hydrazine (triphenylphosphine) palladium (0) (0.138 mg) , 0.119 mmol) and anhydrous THF (2.2 mL) in a stirred mixture. The reaction vessel was sealed and the mixture was heated to 80 °C. After 6 hours at 80 deg.] C, the vessel was cooled to room temperature and carefully add 1 N aqueous HCl (10mL). Diluted with water (15 mL) and EtOAc (3. The combined organic extracts were washed with EtOAc EtOAc mjjjjjjjj ^{ES / MS m / z (35} Cl / 37 Cl) 434.2 / 436.2 [M + H] +.

Preparation 60 1-[(3aR,4R,6S,6aR)-2,2-dimethyl-4-(4-methylpyrrolo[2,3-d]pyrimidin-7-yl)-3a,4,6, Diastereomer of 6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-(3,4-dichlorophenyl)ethanol

A solution of 2.0 M Al(CH ₃ ) ₃ in toluene (0.57 g, 1.41 mmol) was added to 1-[(3aR,4R,6S,6aR)-4-(4-chloropyrrolo[2] at room temperature. ,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxole- Diastereomer of 6-yl]-1-(3,4-dichlorophenyl)ethanol (171.4 mg, 0.354 mmol), hydrazine (triphenylphosphine) palladium (0) (0.082 g, 0.071 mmol) In a mixture of 1,4-dioxane (4.4 mL). The mixture was heated at 80 ° C for 8 hours. The mixture was cooled to rt and was quenched with EtOAc EtOAc. EtOAc (40 mL) was added and EtOAc (EtOAc) The organic extracts were combined and washed with a saturated aqueous solution of sodium bicarbonate and saturated aqueous sodium chloride. The organic extract was dried over MgSO.sub.4, filtered and evaporated. Via a silicone gel with 25-40% of 10% 7 N NH in MeOH in the ₃ / MTBE of 9: 1 mixture of / hexane gradient eluting, the resulting residue was purified to give the title compound (125.9mg, 77% yield rate). ^{ES / MS m / z (35} Cl / 37 Cl) 451/453 [M + H] +.

Preparation 61 (R)-[(3aR,4R,6R,6aR)-2,2-dimethyl-4-(4-methylpyrrolo[2,3-d]pyrimidin-7-yl)-3a,4, 6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methanol

To a high pressure reaction vessel under a N ₂ atmosphere, a solution of 2.0 M Al(CH ₃ ) ₃ in toluene (120 μL, 0.24 mmol) was added to (R)-[(3aR,4R,6R,6aR)-4- (4-Chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3] M-dioxol-6-yl]-(4-chlorophenyl)methanol (0.50 g, 1.15 mmol), hydrazine (triphenylphosphine) palladium (0) (0.266 g, 0.230 mmol) and anhydrous THF (13.8 mL) in a stirred mixture. The reaction vessel was sealed and heated to 70 °C. After 6 hours at 70 ° C, it was cooled to room temperature and was carefully quenched with 1 N aqueous HCI (15 mL). Diluted with water (15 mL) and EtOAc (3. The combined organic extracts were washed with EtOAc EtOAc EtOAc. The residue was purified by EtOAc EtOAc EtOAcEtOAc ^{ES / MS m / z (35} Cl / 37 Cl) 416.0 / 418.0 [M + H] +.

Example 1 (2R,3R,4S,5R)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[(R)-(4-chlorophenyl)-hydroxy- Methyl]tetrahydrofuran-3,4-diol

(R)-[(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl at 0 °C -3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methanol (6.78 g, 13.0 mmol ) Dissolved in TFA (90 mL). Water (9 mL) was added and the mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure and the residue was taken crystalljjjjjjjj The mixture was cooled to 0 ° C and aqueous ammonium hydroxide (28 wt%) was added dropwise to pH~10. The solvent was removed under reduced pressure to give a crude mixture. Purification by silica gel chromatography eluting with a gradient of 0-2% MeOH /EtOAc to afford residue. The residue was dissolved in EtOAc (50 mL)EtOAc. The resulting pale glassy solid was added to water (ca. 50 mL) and the mixture was stirred at room temperature overnight. The resulting white solid was filtered and washed water <RTI ID=0.0> The title compound (3.24 g, 66% yield). ES/MS m/z ( ³⁵ Cl/ ³⁷ Cl) 377.05/379.05 [M+H] ⁺ . ¹ H NMR (300 MHz, d ₆ -DMSO) δ 4.01 (d, J = 4.0 Hz, 2H), 4.63 (dd, J = 5.3, 7.5 Hz, 1H), 4.81 (d, J = 4.0 Hz, 1H), 5.01 (bs, 1H), 5.20 (bs, 1H), 5.91 (d, J = 7.7 Hz, 1H), 6.59 (d, J = 3.7 Hz, 1H), 6.69 (bs, 1H), 7.11 (bs, 2H) ), 7.32 - 7.45 (m, 5H), 8.05 (s, 1H).

Alternative Preparation A Example 1, Crystalline Form I

(R)-[(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl at 0 °C -3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methanol (15.38 g, 0.037 mol) It was slurried in 2-propanol (50 mL). A solution of 4.99 M of 2-propanol HCl (200 mL, 1000 mmol) and water (4 mL) was added and stirred at room temperature for 18 hr. A solution of an additional 4.99 M of 2-propanol HCl (25 mL) was added and the mixture was stirred at room temperature for 24 hours. The mixture was heated at 40 ° C for 2 hours. Concentration under reduced pressure and EtOH (50 mL) and water (50 mL) It was cooled to 0 ° C and an aqueous NH ₄ OH solution (28% yield) was added dropwise to adjust pH to ~10 and the mixture was stirred at room temperature for 2 hours. Concentration under reduced pressure gave a pale yellow solid. Water (50mL) was added dropwise to the yellow solid, followed by addition of _{NH 4 OH (28%, 2mL} ) to adjust the pH to 9, and the mixture was stirred at room temperature for 3 hours. The resulting precipitate was collected by EtOAcqqqqm

Alternative Preparation B, Example 1 Flow chemistry

The reactor 200mL PTFE (od = 1/8 ") disposed inside the GC oven. (300mL) washed with EtOAc. The temperature of the GC oven set at 55 ℃. Using a syringe pump 1L Teledyne ISCO ^TM to 8.5mL / Min pumped (R)-[(3aR,4R,6R,6aR)-4-(4-aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a ,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methanol (186.0 g, 01.57 mmol) 5:2:2 EtOH: a solution of MeOH:EtOAc (1674 mL), which was combined with a 6 N aqueous HCl solution in a T-mixer. The mixture was pumped through the reactor with a peristaltic pump at 1.5 mL/min ( The total residence time was given for 20 minutes. After the feed solution was depleted, the reactor was washed with a 5:1 mixture of EtOAc: 6 N aqueous HCl (400 mL) at 10 mL/min. The concentrated solution was concentrated under reduced pressure. The resulting aqueous solution was cooled to 10 ° C in an ice/water bath. NH ₄ OH (28%, 2 mL) was added dropwise to adjust to pH ~ 10, and the mixture was cooled and stirred at 10 ° C for 35 hours. It was washed with water (100 mL) and dried over EtOAc.

The X-ray powder diffraction pattern of the crystalline solid was obtained on a Bruker D4 Endeavor X-ray powder diffractometer equipped with a CuKa source (λ = 1.54060 Å) and a Vantec detector operating at 35 kV and 50 mA. The sample was scanned at 2θ between 4 and 40°, wherein the step of 2θ was 0.0087° and the scan rate was 0.5 sec/step, and the divergence was 0.6 mm. The fixed anti-scattering property was 5.28mm and the detector slit is 9.5mm. The dry powder was loaded onto a quartz sample holder and a smooth surface was obtained using a glass slide. It is well known in the art of crystallography that for any given crystalline form, the relative intensity of the diffraction peaks may vary due to the preferred orientation resulting from factors such as crystal morphology and habit. In the presence of a preferred orientation, the peak intensity changes, but the characteristic peak position of the polymorph does not change. See, for example, The U.S. Pharmacopeia 35-National Formulary 30, Chapter <941> Characterization of crystalline and partially crystalline solids by X-ray powder diffraction (XRPD) official December 1, 2012 - May 1, 2013. In addition, it is well known in crystallography that the peak position may vary slightly for any given crystalline form. For example, the peak position may be displaced due to temperature or humidity changes as the sample is analyzed, sample displacement, or the presence or absence of an internal standard. In the context of the present invention, a change in the peak position of ± 0.2 of 2θ will take into account such potential changes without prejudice to the unambiguous identification of the specified crystalline form. The crystalline form can be confirmed based on a distinct peak (in degrees 2θ), usually any unique combination of more significant peaks. The diffraction pattern of the crystalline form collected at ambient temperature and relative humidity is adjusted based on the NIST 675 standard peak of 8.84 and 26.76 degrees 2θ.

Crystal form 1

The prepared sample of Form I of Example A was replaced by an X-ray diffraction pattern using CuKa radiation having a diffraction peak (2-theta value) as described in Table 1 below. Specifically, the pattern contains a peak at 25.1° and one or more peaks selected from the group consisting of 17.1°, 13.6°, 20.5°, 24.0°, and 14.5°; wherein the diffraction angle tolerance is 0.2 degrees.

Example 2 (2R,3R,4S,5R)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5[(R)-hydroxy(phenyl)methyl]tetrahydrofuran-3 , 4-diol

Stirring (R)-[(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl at room temperature -3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-phenyl-methanol (0.293 g, 0.766 mmol), 4 N A solution of HCl in 1,4-dioxane (9.6 mL) and water (3 drops). After 45 minutes at room temperature, the solution was concentrated under reduced pressure. The residue was purified by EtOAc (EtOAc)EtOAc ES/MS m/z 343.0 [M+H] ⁺ . _{^{1 H NMR (400MHz, d 6}} -DMSO) δ 4.04-4.06 (m, 2H), 4.55-4.67 (m, 1H), 4.81 (t, J = 3.5Hz, 1H), 4.95 (d, J = 3.9Hz , 1H), 5.19 (d, J = 7.1 Hz, 1H), 5.92 (d, J = 7.9 Hz, 1H), 6.60 (t, J = 3.4 Hz, 2H), 7.11 - 7.13 (m, 2H), 7.24 (t, J = 7.2 Hz, 1H), 7.32 - 7.35 (m, 3H), 7.42 (d, J = 7.3 Hz, 2H), 8.06 (s, 1H).

Example 3 (2R,3R,4S,5S)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[(1R)-1-(4-chlorophenyl)- 1-hydroxy-ethyl]tetrahydrofuran-3,4-diol

(1R)-1-[(3aR,4R,6S,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a ,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-(4-chlorophenyl)ethanol (10.3 g, 23.9 mmol Dissolved in 2-propanol (100 mL) and stirred at 0 °C. A solution of 4.99 M HCl in 2-propanol (200 mL, 1000 mmol) and water (2 mL) was added dropwise and the mixture was stirred at room temperature for 6 hr. The solvent was removed under reduced pressure to give a residue. The residue was dissolved in EtOH (50 mL), the mixture was cooled to 0 &lt;0&gt;C, and aqueous ammonium hydroxide (28 wt% yield) was added dropwise to pH~10. Concentration under reduced pressure gave a white glassy solid. The solid was added to water (ca. 40 mL) and the mixture was stirred at room temperature for 4 hr. The resulting white solid was filtered,jjjjjjjjjj ^{ES / MS m / z (35} Cl / 37 Cl) 391.10 / 393.10 [M + H] +. _{^{1 H NMR (300MHz, d 6}} -DMSO) δ 1.40 (s, 3H), 3.65-3.67 (m, 1H), 4.13 (s, 1H), 4.61-4.68 (m, 1H), 4.78 (d, J = 3.7 Hz, 1H), 5.12 (d, J = 7.0 Hz, 1H), 5.81 (d, J = 8.1 Hz, 1H), 6.59 (d, J = 3.7 Hz, 1H), 7.10 (bs, 1H), 7.19 (bs, 2H), 7.32 (d, J = 3.3 Hz, 1H), 7.40 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.8 Hz, 2H), 8.07 (s, 1H).

Example 4

(2R,3R,4S,5R)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[(R)-[4-chloro-3-(cyclopropane Diastereomers of oxy)phenyl]-hydroxy-methyl]tetrahydrofuran-3,4-diol

A 15 mL Teflon-coated SS reactor (od = 1/8") was placed in a Vapourtec E-series apparatus. A back pressure regulator (4-5 bar) was applied to the outlet of the reaction system and the temperature was set and The temperature was set at 82 ° C. (R)-[(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidine-7- was pumped at 1.1 mL/min. -2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-[4-chloro 3- (cyclopropyloxy) phenyl] methanol the non diastereomers (32.1g, 60.1mmol) in EtOH (317mL) in the solution, and the solution in the mixer T- and 4 N HCl The aqueous stream was combined and the mixture was pumped through the reactor at 0.288 mL/min at 82 ° C (giving a total residence time of 10 minutes). After depleting the feed solution, the mixture was treated with EtOH / 4 N HCl ( 400 mL) at 0.29mL / min the reactor was flushed, Darco KB ^® (100 mesh with activated carbon; wet powder, 13g) treating the crude reaction mixture, and filtered through a short pad of diatomaceous earth with activated carbon Darco KB ^® (100. Mesh; wet powder, 26g) was repeatedly treated and filtered through diatomaceous earth. Reduce the organic solvent of the collected solution by 1⁄4 volume under vacuum, add dropwise Water was added (150 mL) and aqueous NH ₄ OH (28%, 40mL), the pH was adjusted to ~ 10, and extracted with EtOAc (2 × 300mL). The combined organic extracts were washed with saturated aqueous sodium chloride, dried over magnesium Drying, filtration and concentrating the filtrate <RTI ID=0.0></RTI> to EtOAcjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj . product (7.89 g) Both residues were combined to 1:10 MeOH: EtOAc (300mL), and the solvent was evaporated under reduced pressure to give the title compound (17.45g; 67% yield) .ES / MS m / z ( ³⁵ Cl/ ³⁷ Cl) 433.0/435.0 [M+H] ^{+ 1} H NMR (300 MHz, d _{6 -} DMSO) δ 0.73-0.81 (m, 4H), 3.77-3.83 (m, 1H), 4.00 ( d, J = 4.6 Hz, 1H), 4.04-4.09 (m, 1H), 4.67-4.73 (m, 1H), 4.81 (t, J = 4.0 Hz, 1H), 5.04 (d, J = 3.8 Hz, 1H) ), 5.21 (d, J = 7.3 Hz, 1H), 5.91 (d, J = 7.9 Hz, 1H), 6.58 (d, J = 3.6 Hz, 1H), 6.65 (d, J = 3.4 Hz, 1H), 7.01 (dd, J=1.5, 8.2 Hz, 1H), 7.10 (bs, 2H), 7.35 (d, J = 1.6 Hz, 1H), 7.43 (d, J = 1.4 Hz, 1H), 8.06 (s, 1H) ).

Example 5 (2R,3R,4S,5R)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[hydroxy-[4-(trifluoromethyl) Diastereomer of phenyl]methyl]tetrahydrofuran-3,4-diol

[(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a - diastereomer of tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-[4-(trifluoromethyl)phenyl]methanol (13.5 g, 27.0 mmol) was dissolved in 2-propanol (50 mL) and stirred at 0 °C. A solution of 4.99 M HCl in 2-propanol (200 mL, 0.998 mol) and water (2 mL) was added dropwise to the mixture and stirred at room temperature for 3 hr. The solvent was removed under reduced pressure to give a residue. The residue was dissolved in EtOH (50 mL) and the mixture was cooled to 0. An aqueous ammonium hydroxide solution (28 wt%) was added dropwise to pH ~ 10, and the mixture was stirred for 10 minutes. The solvent was removed under reduced pressure to give a white white solid. Purification by silica gel chromatography eluting with a gradient of 80-100% EtOAc/hexanes afforded a foam. The foam was suspended in water (300 mL), and the mixture was stirred at room temperature for 1 hour. The resulting white solid was filtered,jjjjjjjjjj ES/MS m/z 411.10 [M+H] ⁺ . _{^{1 H NMR (300MHz, d 6}} -DMSO) δ 4.02-4.07 (m, 2H), 4.65 (td, J = 7.4,5.1Hz, 1H), 4.92 (t, J = 3.6Hz, 1H), 5.02 (d , J = 3.8 Hz, 1H), 5.22 (d, J = 7.1 Hz, 1H), 5.92 (d, J = 8.0 Hz, 1H), 6.60 (d, J = 3.6 Hz, 1H), 6.81 (d, J = 3.3 Hz, H), 7.11 (s, 2H), 7.32 (d, J = 3.8 Hz, 1H), 7.71 - 7.63 (m, 4H), 8.06 (s, 1H).

Example 6 (2R,3R,4S,5R)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[(3-chlorophenyl)-hydroxy-methyl]tetrahydrofuran -3,4-diol diastereomer

Mix [(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a - diastereomer of tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3-chlorophenyl)methanol (0.052 g, 0.124 mmol) 4 N HCl in 1,4-dioxane (5 mL) and MeOH (5 mL). The mixture was stirred at room temperature. After 1 hour at room temperature, the solution was concentrated under a stream of N ₂ to remove solvent. Dissolved in water (5 mL) and EtOAc (2 mL). A 1 N aqueous solution of NaOH was added until the solution was basic and extracted with DCM (4×3 mL). The organic extracts were combined, dried over sodium sulfate, filtered and evaporated. Purification by chromatography eluting with EtOAc EtOAc EtOAc (EtOAc) ^{ES / MS m / z (35} Cl / 37 Cl) 377.0 / 379.0 [M + H] +. _{^{1 H NMR (400MHz, d 6}} -DMSO) δ 3.37 (bs, 1H), 4.04-4.08 (m, 2H), 4.63-4.69 (m, 1H), 4.85-4.88 (m, 1H), 5.07 (d, J=3.6 Hz, 1H), 5.26 (d, J=7.0 Hz, 1H), 5.96 (d, J=8.1 Hz, 1H), 6.64 (d, J=3.5 Hz, 1H), 6.76-6.74 (m, 1H), 7.15-7.18 (bs, 1H), 7.33-7.42 (m, 4H), 7.50-7.52 (m, 1H), 8.09 (s, 1H).

Example 7 (2R,3R,4S,5R)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[(4-chloro-2-fluoro-phenyl)-hydroxyl -Methyl] diastereomer of tetrahydrofuran-3,4-diol hydrochloride

Stir [(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4 at room temperature , diastereomeric, 6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chloro-2-fluoro-phenyl)methanol A solution of the org. (0.067 g, 0.154 mmol), 4 N EtOAc in EtOAc (EtOAc) After 1 hour at room temperature, the solution was concentrated under a stream of N ₂ to remove solvent. The residue was triturated with EtOAc (EtOAc) ^{ES / MS m / z (35} Cl / 37 Cl) 395/397 [M + H] +. _{^{1 H NMR (400MHz, d 6}} -DMSO) δ 3.97-4.00 (m, 1H), 4.09-4.11 (m, 1H), 4.44-4.49 (m, 1H), 4.92-4.96 (m, 1H), 6.06- 6.09 (m, 1H), 6.98-7.00 (m, 1H), 7.26-7.28 (m, 1H), 7.32-7.35 (m, 1H), 7.51-7.56 (m, 1H), 7.65-7.66 (m, 1H) ), 8.35 (s, 1H).

Example 8 (2R,3R,4S,5R)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[(2-fluorophenyl)-hydroxy-methyl]tetrahydrofuran -3,4-diol diastereomer

Evacuation (2R,3R,4S,5R)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[(4-chloro) in a standard container under reduced pressure Diastereomer of 2-fluoro-phenyl)-hydroxy-methyl]tetrahydrofuran-3,4-diol hydrochloride (0.036 g, 0.083 mmol), 10% Pd/C (0.010 mg, 0.009) Mixture of mmol) and EtOH (2.0 mL). The vessel was pressurized to 10 psi with hydrogen. After stirring at 10 psi for 24 hours, triethylamine (0.25 mg, 0.25 mmol) and an additional 10% Pd/C (0.010 mg, 0.009 mmol) were added. The vessel was evacuated and pressurized to 10 psi with hydrogen. Stir for a further 24 hours at room temperature. The resulting mixture was filtered through celite and concentrated under reduced pressure. Via reverse phase high pressure chromatography (PHENOMENEX ^® GEMINI ^® -NX), over 20 minutes, to a mixture of 5-45% ACN in 5% MeOH / 10mM of ammonium bicarbonate (pH ~ 10) eluting with a gradient, the resulting residue was purified The title compound (0.010 g, 33% yield). ES/MS m/z 361[M+H] ⁺ . ¹ H NMR (400 MHz, d ₆ -DMSO) δ 4.01-4.03 (m, 2H), 4.63-4.64 (m, 1H), 4.97-5.01 (m, 1H), 5.19-5.21 (m, 1H), 5.89 - 5.93 (m, 1H), 6.56-6.57 (m, 1H), 6.85-6.89 (m, 1H), 7.31-7.32 (m, 5H), 7.60-7.61 (m, 1H), 8.02 (s, 1H) .

Example 9 ((2R,3R,4S,5R)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[(3-ethylphenyl)-hydroxy-methyl Diastereomer of tetrahydrofuran-3,4-diol hydrochloride

Stir [(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4 at room temperature , 6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3-ethylphenyl)methanol diastereomer (0.240 g, 0.585 mmol), 4 N HCl in 1,4-dioxane (5.0 mL) and MeOH (5.0 mL). After 1 hour at room temperature, the solution was concentrated under a stream of N ₂ to remove solvent. The residue was triturated with EtOAc (EtOAc) ES/MS m/z 371 [M+H] ⁺ . ¹ H NMR (400 MHz, d ₆ -DMSO) δ 1.12 (t, J = 7.6 Hz, 3H), 2.51-2.57 (q, J = 7.6 Hz, 2H), 3.98-4.02 (m, 1H), 4.08 - 4.11 (m, 1H), 4.546-4.50 (m, 1H), 4.69-4.72 (m, 1H), 6.06 (d, J = 7.7 Hz, 1H), 6.97 (d, J = 3.7 Hz, 1H), 7.01 7.06 (m, 1H), 7.14 - 7.27 (m, 4H), 7.68 (d, J = 3.6 Hz, 1H), 8.35 (s, 1H).

Example 10 4-[[(2R,3S,4R,5R)-5-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl] -dihydroxy-methyl]benzonitrile diastereomer

TFA (3.5 mL) and water (0.5 mL) were combined and cooled to 0 °C. Add this to 4-[[(3aR,4R,6R,6aR)-4-(4-aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a , diastereomer of 4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-hydroxy-methyl]benzonitrile ( In 0.50 g, 1.24 mmol), and stirred at 0 °C. After 1 hour, it was concentrated under reduced pressure and the obtained solid was dissolved in MeOH. SILICYCLE ^® with MeOH via silica column carbonate (70mL, 5g) eluting. The title compound (0.47 g, 100% yield). ES/MS m/z 368 [M+H] ⁺ . ¹ H NMR (400 MHz, d ₆ -DMSO) δ 3.33-3.41 ( _m , 2H), 4.04 (d, J = 4.2 Hz, 2H), 4.61-4.65 (m, 1H), 4.91 (d, J = 4.3 Hz) , 1H), 5.06 (d, J = 2.4 Hz, 1H), 5.22 - 5.26 (m, 1H), 5.94 (d, J = 7.8 Hz, 1H), 6.65 (d, J = 3.6 Hz, 1H), 6.71 -6.79 (m, 1H), 7.31 - 7.35 (m, 2H), 7.39 (d, J = 3.6 Hz, 1H), 7.62 (d, J = 8.3 Hz, 2H), 7.80 (d, J = 8.3 Hz, 2H), 8.10 (s, 1H).

Example 11 (2R,3R,4S,5R)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[(3,4-difluorophenyl)-hydroxy-methyl Diastereomer of tetrahydrofuran-3,4-diol

Mix [(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4 at 0 °C , 6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3,4-difluorophenyl)methanol diastereomer (694mg, 1.66mmol), 1,4- dioxane (6mL), water (2mL), 4 M in 1,4-dioxane of HCl (3mL, 12 mmol). The mixture was stirred for 18 hours, add additional 4 M in 1,4-dioxane of HCl (4mL, 16mmol). The mixture was stirred at room temperature for additional 18 hours. HCl (31 wt% in water, 0.5 mL) was added. The mixture was stirred at room temperature for 4 hours. The mixture was cooled at 0 ° C and a saturated NaHCO ₃ solution was slowly added until a pH ~ 7 was achieved. Extract with 2-4% MeOH / DCM. The organic extract was dried over MgSO.sub.4, filtered and evaporated. The residue was purified by EtOAc EtOAc EtOAc. ES/MS m/z 379 [M+H] ⁺ . _{^{1 H NMR (400MHz, d 6}} -DMSO) δ 4.02-4.04 (m, 2H), 4.63 (td, J = 7.3,5.2Hz, 1H), 4.82 (t, J = 3.9Hz, 1H), 5.03 (d , J = 4.0 Hz, 1H), 5.22 (d, J = 7.1 Hz, 1H), 5.92 (d, J = 7.8 Hz, 1H), 6.61 (d, J = 3.6 Hz, 1H), 6.73 (d, J = 3.6 Hz, 1H), 7.11 (s, 2H), 7.27 (dd, J = 4.0, 8.2 Hz, 1H), 7.34 (d, J = 3.6 Hz, 1H), 7.39-7.46 (m, 2H), 8.06 (s, 1H).

Example 12 (2R,3R,4S,5R)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[(3,4-dichlorophenyl)-hydroxy-methyl Diastereomer of tetrahydrofuran-3,4-diol

Mix [(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4 at room temperature , 6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(3,4-dichlorophenyl)methanol diastereomer (180.0 mg, 0.398 mmol), water (0.20 mL) and TFA (2.0 mL). After 45 minutes, the mixture was concentrated under reduced pressure and the obtained residue was dissolved in MeOH. SILICYCLE ^® with MeOH via silica column carbonate (70mL, 5g) eluting mixture. Appropriate fractions were collected and concentrated in vacuo to give a crude residue. With 10% NH ₃ / MeOH in CHCl ₃ to the mixture via silica gel chromatography eluting gradient, the residue was purified to give the title compound as a diastereomeric forms of (98.3mg, 60% yield). ^{ES / MS m / z (35} Cl / 37 Cl) 411/413 [M + H] +. _{^{1 H NMR (400MHz, d 6}} -DMSO) δ 4.01-4.03 (m, 2H), 4.62 (td, J = 7.3,5.0Hz, 1H), 4.84 (t, J = 4.0Hz, 1H), 5.05 (d , J = 3.9 Hz, 1H), 5.22 (d, J = 7.1 Hz, 1H), 5.92 (d, J = 7.8 Hz, 1H), 6.60 (d, J = 3.6 Hz, 1H), 6.77 (d, J = 3.6 Hz, 1H), 7.11 (s, 2H), 7.34 (d, J = 3.6 Hz, 1H), 7.41 (dd, J = 1.8, 8.4 Hz, 1H), 7.59 (d, J = 8.3 Hz, 1H) ), 7.65 (d, J = 1.8 Hz, 1H), 8.06 (s, 1H).

Example 13 (2R,3R,4S,5R)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[hydroxy(p-tolyl)methyl]tetrahydrofuran-3,4 - diastereomers of diols

Mix [(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4 at room temperature , 6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(p-tolyl)methanol diastereomer (115 mg, 0.290 mmol ), water (0.15 mL) and TFA (1.5 mL). After 90 minutes, it was concentrated under reduced pressure and the obtained solid was dissolved in MeOH. SILICYCLE ^® with MeOH via silica column carbonate (70mL, 5g) eluting. The appropriate fractions were collected and evaporated to dryness under reduced pressure to give a residue. Via a silicone gel with 5-15% of 10% 7 N NH ₃ / MeOH mixture of / DCM / gradient eluting, the residue was purified to give the title compound as a diastereomeric forms of (50.2mg, 49% Yield). ES/MS m/z 357 [M+H] ⁺ . _{^{1 H NMR (400MHz, d 6}} -DMSO) δ 2.29 (s, H), 4.62-4.67 (m, 1H), 4.77 (t, J = 3.3Hz, 1H), 4.93 (d, J = 3.8Hz, 1H ), 5.18 (d, J = 7.1 Hz, 1H), 5.90 (d, J = 7.9 Hz, 1H), 6.58 (dd, J = 3.4, 12.3 Hz, 2H), 7.11-7.15 (m, 3H), 7.29 -7.33 (m, 3H), 8.06 (s, 1H).

Example 14 (2R,3R,4S,5R)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[(4-fluorophenyl)-hydroxy-methyl]tetrahydrofuran -3,4-diol diastereomer

Mix [(3aR,4R,6R,6aR)-4-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a - a diastereomer of tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-fluorophenyl)methanol (158 mg, 0.394 mmol), 1,4-dioxane (3 mL), water (0.75 mL) and 4 M HCl in 1,4-dioxane (3mL, 12mmol) in the solution, and the mixture was stirred at 0 ℃ for 5 minutes and then at room Stir for 1 hour at room temperature. Concentrated under reduced pressure, then EtOAc (30 mL)EtOAc. This procedure was repeated with the addition of EtOAc and twice more concentrated. The residue was purified by EtOAc EtOAc EtOAc EtOAc EtOAc ES/MS m/z 361[M+H] ⁺ . ¹ H NMR (400 MHz, d _{6 -} DMSO) δ 4.03 (dd, J=4.3, 10.6 Hz, 2H), 4.63 (td, J = 7.3, 5.2 Hz, 1H), 4.81 (t, J = 3.7 Hz, 1H) ), 4.99 (d, J = 3.9 Hz, 1H), 5.20 (d, J = 7.1 Hz, 1H), 5.92 (d, J = 7.8 Hz, 1H), 6.61 (dd, J = 3.6, 9.5 Hz, 2H) ), 7.11-7.17 (m, 4H), 7.32 (d, J = 3.6 Hz, 1H), 7.45 (dd, J = 5.8, 8.5 Hz, 2H), 8.06 (s, 1H).

Example 15 (2R,3R,4S,5S)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[1-(4-chlorophenyl)-1-hydroxy- Diastereomer of propyl]tetrahydrofuran-3,4-diol

[(3aR,4R,6S,6aS)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4,6,6a- Diastereomer of tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methanone (426.9 mg, 0.983 mmol) Dissolved in THF (5 mL) and the mixture was cooled to 0 °C. A solution of 1.0 M ethylmagnesium bromide in THF (1.97 mL, 1.97 mmol) was added over 1 min. Stir for 15 minutes and warm to room temperature. After 1 hour, by the addition of 1 N aqueous HCl (2.48mL) and quenched. The reaction mixture was diluted with DCM (10 mL). The layers were separated and aqueous was extracted with DCM (5 mL). The organic extracts were combined and evaporated under reduced pressure. The residue was dissolved in _{7 M NH 3 / MeOH (10mL} , 70mmol) and heated at 100 deg.] C in a microwave reactor for 6 hours. The resulting mixture was evaporated at 50 ° C under a stream of N ₂ . The resulting residue was dissolved in 4 N in 1,4-dioxane (1.29mL, 49.2mmol) in of HCl, and the addition of water (35μL, 1.97mmol). The mixture was stirred at room temperature for 1.5 hours, and then the mixture was concentrated under reduced pressure. Via reverse phase high pressure liquid chromatography (PHENOMENEX ^® GEMINI ^® -NX), with 13-48% ACN in a mixture of 5% MeOH / 10mM of ammonium bicarbonate (pH ~ 10) in a gradient of eluting, the resulting residue was purified, The title compound was obtained as a diastereomer (97.8 mg, 25% yield). ^{ES / MS m / z (35} Cl / 37 Cl) 405.1 / 407.2 [M + H] +. ¹ H NMR (400 MHz, d ₆ -DMSO) δ 0.54 (t, J = 7.4 Hz, 3H), 1.75-1.92 (m, 2H), 3.63 (d, J = 5.0 Hz, 1H), 4.22 (s, 1H) ), 4.69 (dd, J = 5.3, 7.6 Hz, 1H), 4.78-4.82 (m, 1H), 5.14-5.20 (m, 1H), 5.75 (d, J = 8.1 Hz, 1H), 6.59 (d, J = 3.5 Hz, 1H), 7.11 (s, 1H), 7.23 (s, 1H), 7.31 (d, J = 3.6 Hz, 1H), 7.42 (d, J = 8.7 Hz, 2H), 7.53 - 7.56 ( m, 2H), 8.08 (s, 1H).

Example 16 (2R,3R,4S,5S)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[1-(3,4-dichlorophenyl)-1 -Hydroxy-ethyl]tetrahydrofuran-3,4-diol diastereomer

Combining 1-[(3aR,4R,6S,6aR)-4-(4-aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a at room temperature , 4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-(3,4-dichlorophenyl)ethanol (101.2 mg, 0.217 mmol), water (0.1 mL) and TFA (1.0 mL). After stirring for 1 hour, it was concentrated under reduced pressure and the obtained solid was dissolved in MeOH. SILICYCLE ^® with MeOH via silica column carbonate (70mL, 5g) eluting. The appropriate fractions were collected to give a residue. In MeOH / MeOH / chloroform (by volume 1: 1: 2) with 5-30% 10% 7 N NH ₃ in the mixture was chromatographed through silica gel / CHCl ₃ gradient of eluting, the resulting residue was purified to afford The title compound (81.7 mg, 88% yield). ^{ES / MS m / z (35} Cl / 37 Cl) 426/428 [M + H] +. _{^{1 H NMR (400MHz, d 6}} -DMSO) δ 1.42 (s, 3H), 3.66 (t, J = 4.4Hz, 1H), 4.15 (s, 1H), 4.56-4.65 (m, 1H), 4.85 (d , J = 3.7 Hz, 1H), 5.14 (d, J = 7.2 Hz, 1H), 5.83 (d, J = 8.0 Hz, 1H), 6.61 (d, J = 3.6 Hz, 1H), 7.20 (s, 2H) ), 7.27 (s, 1H), 7.33 (d, J = 3.5 Hz, 1H), 7.55 (dd, J = 2.0, 8.5 Hz, 1H), 7.62 (d, J = 8.5 Hz, 1H), 7.81 (d) , J = 2.0 Hz, 1H), 8.08 (s, 1H).

Example 17 (2R,3R,4S,5S)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[1-(2-fluorophenyl)-1-hydroxy- Diastereomer of ethyl]tetrahydrofuran-3,4-diol

TFA (4 mL) and water (1 mL) were combined and cooled to 0. Add this to 1-[(3aR,4R,6S,6aR)-4-(4-aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a, Diastereomer of 4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-(2-fluorophenyl)ethanol (0.341 g, 0.823 mmol), and stirred at 0 °C. After 30 minutes, it was concentrated under reduced pressure and the obtained solid was dissolved in MeOH. SILICYCLE ^® with MeOH via silica column carbonate (70mL, 5g) eluting. Appropriate fractions were collected and chromatographed through silica gel with 0-100% of 10% 7 N NH ₃ / MeOH mixture solvent gradient of DCM / from further purified, to obtain the form of the diastereomers of the title compound (0.271 g , 88% yield). ES / MS m / z 375.0 [ M + H] +. _{^{1 H NMR (400MHz, d 6}} -DMSO) δ 1.49 (s, 3H), 3.33 (s, 2H), 3.64 (t, J = 4.4Hz, 1H), 4.32 (s, 1H), 4.68 (td, J = 7.5, 5.2 Hz, 1H), 4.80 (d, J = 3.7 Hz, 1H), 5.13 (d, J = 7.1 Hz, 1H), 5.83 (d, J = 8.1 Hz, 1H), 6.61 (d, J = 3.5 Hz, 1H), 7.15-7.27 (m, 4H), 7.31 - 7.35 (m, 3H), 7.81 (td, J = 8.0, 1.6 Hz, 1H), 8.09 (s, 1H).

Example 18 (2R,3R,4S,5S)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[1-hydroxy-1-[2-(trifluoromethyl) Diastereomer of phenyl]ethyl]tetrahydrofuran-3,4-diol

TFA (3 mL) and water (1 mL) were combined and cooled to 0 °C. This mixture was added to 1-[(3aR,4R,6S,6aR)-4-(4-aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a ,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-[2-(trifluoromethyl)phenyl]ethanol The diastereomer (0.137 g, 0.295 mmol) was stirred at 0 °C. After 20 minutes, the reaction mixture was concentrated under reduced pressure and the obtained solid was dissolved in MeOH. SILICYCLE ^® with MeOH via silica column carbonate (70mL, 5g) eluting solution. Appropriate fractions were collected and chromatographed through silica gel with 0-100% of 10% 7 N NH ₃ / MeOH mixture solvent gradient of DCM / from further purified, to obtain the form of the diastereomers of the title compound (0.109 g , 87% yield). ES/MS m/z 425.0 [M+H] ⁺ . _{^{1 H NMR (400MHz, d 6}} -DMSO) δ 1.52 (s, 3H), 3.76 (t, J = 4.4Hz, 1H), 4.38 (s, 1H), 4.64 (dd, J = 7.6,12.8Hz, 1H ), 4.80 (d, J = 3.7 Hz, 1H), 5.14 (d, J = 7.2 Hz, 1H), 5.86 (d, J = 8.1 Hz, 1H), 6.61 (d, J = 3.5 Hz, 1H), 6.93 (s, 1H), 7.17 (s, 2H), 7.38 (d, J = 3.6 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.65 (t, J = 7.5 Hz, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 8.06 (s, 1H).

Example 19 (2R,3R,4S,5S)-2-(4-Aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[1-hydroxy-1-[4-(trifluoromethyl) Diastereomer of phenyl]ethyl]tetrahydrofuran-3,4-diol

TFA (2 mL) and water (0.5 mL) were combined and cooled to 0 °C. Add this to 1-[(3aR,4R,6S,6aR)-4-(4-aminopyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a, 4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-[4-(trifluoromethyl)phenyl]ethanol The enantiomer (0.136 g, 0.292 mmol) was stirred at 0 °C. After 45 minutes, it was concentrated under reduced pressure and the obtained solid was dissolved in MeOH. SILICYCLE ^® with MeOH via silica column carbonate (70mL, 5g) eluting. Appropriate fractions were collected and chromatographed through silica gel with 10-25% of 10% 7 N NH ₃ / MeOH mixture solvent gradient of DCM / from further purified, to obtain the form of the diastereomers of the title compound (0.095 g , 76% yield). ES/MS m/z 425.0 [M+H] ⁺ . ¹ H NMR (400 MHz, d ₆ -DMSO) δ 1.44 (s, 3H), 3.66 (t,J=4.3 Hz, 1H), 4.20 (s, 1H), 4.67 (td, J = 7.5, 5.2 Hz, 1H) ), 4.81 (d, J = 3.7 Hz, 1H), 5.14 (d, J = 7.2 Hz, 1H), 5.83 (d, J = 8.1 Hz, 1H), 6.61 (d, J = 3.6 Hz, 1H), 7.21(s, 2H), 7.27(s, 1H), 7.33 (d, J = 3.6 Hz, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.2 Hz, 2H), 8.09 (s, 1H).

Example 20 (2S,3S,4R,5R)-2-[(1R)-(1-(4-Chlorophenyl)-1-hydroxy-ethyl]-5-(4-methylpyrrolo[2,3- d]pyrimidin-7-yl)tetrahydrofuran-3,4-diol

Stirring (1R)-1-[(3aR,4R,6S,6aR)-2,2-dimethyl-4-(4-methylpyrrolo[2,3-d]pyrimidine-7- at room temperature -3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-(4-chlorophenyl)ethanol (0.265 g, 0.616 mmol), a mixture of 4 N EtOAc in EtOAc (EtOAc) After 1 hour at room temperature, the solution was concentrated under a stream of N ₂ to remove solvent. Water (30mL),, and extracted with DCM (3 × 20mL) with saturated aqueous NaHCO ₃ and to the pH ~ 7. The organic extracts were combined, dried over sodium sulfate, filtered and evaporated. The residue was purified by EtOAc EtOAc EtOAc elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 390.02 / 392.2 [M + H] +. _{^{1 H NMR (400MHz, d 6}} -DMSO) δ 1.39 (s, 3H), 2.46-2.48 (m, 5H), 3.30 (s, 1H), 2.65 (s, 3H), 3.68 (t, J = 4.3Hz , 1H), 4.08 (s, 1H), 4.51-4.54 (m, 1H), 4.84 (d, J = 3.7 Hz, 1H), 5.15-5.17 (m, 1H), 6.07 (d, J = 7.9 Hz, 1H), 6.18 (s, 1H), 6.76 (d, J = 3.7 Hz, 1H), 7.37-7.39 (m, 2H), 7.53 - 7.56 (m, 2H), 7.79 (d, J = 3.7 Hz, 1H) ), 8.64 (s, 1H).

Example 21 (2R,3S,4R,5R)-2-[(4-chloro-2-fluoro-phenyl)-hydroxy-methyl]-5-(4-methylpyrrolo[2,3-d]pyrimidine- 7-yl) diastereomer of tetrahydrofuran-3,4-diol

Stir [(3aR,4R,6R,6aR)-2,2-dimethyl-4-(4-methylpyrrolo[2,3-d]pyrimidin-7-yl)-3a,4 at room temperature , diastereoisomerism of 6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chloro-2-fluoro-phenyl)methanol A solution of the org. (0.220 g, 0.507 mmol), 4 N EtOAc in EtOAc (EtOAc) After 1 hour at room temperature, the solution was concentrated under a stream of N ₂ to remove solvent. The residue was partitioned between DCM (25 mL) and water (25 mL). , And extracted with DCM (4 × 20mL) with saturated aqueous NaHCO ₃ and to the pH ~ 7. The organic extracts were combined, dried over sodium sulfate, filtered and evaporated. The residue was purified by EtOAc EtOAc elut elut elut elut elut elut ^{ES / MS m / z (35} Cl / 37 Cl) 394.2 / 396.2 [M + H] +. _{^{1 H NMR (400MHz, d 6}} -DMSO) δ 2.63 (s, 3H), 3.28-3.32 (m, 2H), 3.97-4.02 (m, 1H), 4.08-4.10 (m, 1H), 4.54-4.57 ( m,1H),4.94-4.97(m,1H),5.13-5.16(m,1H),5.26-5.30(m,1H), 6.14(d,J=7.7Hz,1H),6.23(d,J= 4.5 Hz, 1H), 6.75 (d, J = 3.7 Hz, 1H), 7.26 (dd, J = 1.9, 8.3 Hz, 1H), 7.33 (dd, J = 2.0, 10.2 Hz, 1H), 7.54 (t, J = 8.2 Hz, 1H), 7.71 (d, J = 3.9 Hz, 1H), 8.61 (s, 1H).

Example 22 ((2R,3S,4R,5R)-2-[(2-fluorophenyl)-hydroxy-methyl]-5-(4-methylpyrrolo[2,3-d]pyrimidin-7-yl) Diastereomer of tetrahydrofuran-3,4-diol

Evacuation (2R,3S,4R,5R)-2-[(4-chloro-2-fluoro-phenyl)-hydroxy-methyl]-5-(4-methylpyrrole in a standard vessel under reduced pressure [2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diol diastereomer (0.090 g, 0.228 mmol), 10% Pd/C (0.025 g), triethylamine ( A mixture of 0.069 g, 0.68 mmol) and EtOH (4.0 mL). The vessel was pressurized to 10 psi with hydrogen and the resulting mixture was stirred at room temperature for 15 hours. The resulting mixture was filtered through celite and concentrated under reduced pressure. The residue was purified by EtOAc EtOAc EtOAc elut elut elut elut elut ES/MS m/z 360.2 [M+H] ⁺ . _{^{1 H NMR (400MHz, d 6}} -DMSO) δ 2.63 (s, 3H), 4.01-4.04 (m, 1H), 4.08-4.11 (m, 1H), 4.56-4.58 (m, 1H), 4.97-5.00 ( m, 1H), 5.10 (d, J = 4.2 Hz, 1H), 5.26 (d, J = 7.1 Hz, 1H), 6.13-6.18 (m, 2H), 6.75 (d, J = 3.7 Hz, 1H), 7.12-7.14 (m, 1H), 7.18-7.21 (m, 1H), 7.29-7.31 (m, 1H), 7.56-7.57 (m, 1H), 7.71-7.72 (m, 1H), 8.62 (s, 1H) ).

Example 23 (2S,3S,4R,5R)-2-[1-(3,4-Dichlorophenyl)-1-hydroxy-ethyl]-5-(4-methylpyrrolo[2,3-d] Diastereomer of pyrimidine-7-yl)tetrahydrofuran-3,4-diol

Mix 1-[(3aR,4R,6S,6aR)-2,2-dimethyl-4-(4-methylpyrrolo[2,3-d]pyrimidin-7-yl)-3a at room temperature , 4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-1-(3,4-dichlorophenyl)ethanol (122.0 mg, 0.26 mmol), water (0.15 mL) and TFA (l. After 1 hour, it was concentrated under reduced pressure and the obtained solid was dissolved in MeOH. SILICYCLE ^® with MeOH via silica column carbonate (70mL, 5g) eluting. The appropriate fractions were collected and concentrated to dryness under reduced pressure to give a residue. The residue was purified by EtOAc (EtOAc) elut elut elut elut elut . ^{ES / MS m / z (35} Cl / 37 Cl) 426/428 [M + H] +. _{^{1 H NMR (400MHz, d 6}} -DMSO) δ 1.45 (s, 3H), 2.69 (s, 3H), 3.73 (t, J = 4.5Hz, 1H), 4.14 (s, 1H), 4.52 (dd, J = 7.4, 12.7 Hz, 1H), 4.92 (d, J = 4.0 Hz, 1H), 5.19 (d, J = 7.0 Hz, 1H), 6.12 (d, J = 7.8 Hz, 1H), 6.32 (s, 1H) ), 6.80 (d, J = 3.7 Hz, 1H), 7.55 (dd, J = 2.0, 8.5 Hz, 1H), 7.63 (d, J = 8.5 Hz, 1H), 7.80-7.81 (m, 2H), 8.68 (s, 1H).

Example 24 and Example 25 (2R,3S,4R,5R)-2-[(R)-hydroxy(phenyl)methyl]-5-pyrrolo[2,3-d]pyrimidin-7-yl-tetrahydrofuran-3,4-di alcohol

(2R,3S,4R,5R)-2-[(R)-(4-chlorophenyl)-hydroxy-methyl]-5-pyrrolo[2,3-d]pyrimidin-7-yl-tetrahydrofuran- 3,4-diol

(R)-[(3aR,4R,6R,6aR)-4-(4-chloropyrrolo[2,3-d]pyrimidin-7-yl)-2,2-dimethyl-3a,4, 6,6a-Tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methanol (200.0 mg, 0.264 mmol) was dissolved in EtOAc. 30mL). At room temperature (2000kPa / 1mL / min / 70mm Pd / Al 2 O 3 cartridge) was hydrogenated with flow THALESNANO ^TM H-Cube system. The resulting solution was evaporated under reduced pressure, and the resulting residue was dissolved in EtOAc (20mL), and the hydrogenation was repeated with a new Pd / Al ₂ O ₃ cartridge. The resulting solution was evaporated under reduced pressure. The residue was dissolved in 4 N in 1,4-dioxane (5.73mL, 22.9mmol) in of HCl, and water (3 drops). The solution was stirred at room temperature for 45 minutes and evaporated under reduced pressure. With 5-38% ACN in 5% MeOH / mixture (pH ~ 10) eluting with a gradient, the resulting residue was purified by reverse phase high pressure of 10mM ammonium bicarbonate chromatography (PHENOMENEX ^® GEMINI ^® -NX), to give Example 23 And example 24.

Example 24 (2R,3S,4R,5R)-2-[(R)-Hydroxy(phenyl)methyl]-5-pyrrolo[2,3-d]pyrimidin-7-yl-tetrahydrofuran-3,4 -diol (15.0 mg, 9.11% yield). ES/MS m/z 328.1 [M+H)]. ¹ H NMR (400 MHz, d ₆ -DMSO) δ 3.18 (d, J = 5.3 Hz, 3H), 4.05 - 4.15 (m, 3H), 4.62 (dd, J = 5.2, 7.5 Hz, 1H), 4.80 (t , J=4.2 Hz, 1H), 5.10 (s, 1H), 5.22-5.30 (m, 1H), 6.22 (d, J = 7.8 Hz, 1H), 6.74 (d, J = 3.7 Hz, 1H), 7.24 (t, J = 7.2 Hz, 1H), 7.32 (t, J = 7.5 Hz, 2H), 7.41 (d, J = 7.4 Hz, 2H), 7.87 (d, J = 3.7 Hz, 1H), 8.81 (s) , 1H), 9.04 (s, 1H).

Example 25 (2R,3S,4R,5R)-2-[(R)-(4-Chlorophenyl)-hydroxy-methyl]-5-pyrrolo[2,3-d]pyrimidin-7-yl- Tetrahydrofuran-3,4-diol (51.9 mg, 28.8% yield). ES/MS m/z ( ³⁵ Cl/ ³⁷ Cl) 36.1/364.1 [M+H] ⁺ . ¹ H NMR (400 MHz, d ₆ -DMSO) δ 3.18 (d, J = 5.3 Hz, 3H), 4.01 (d, J = 5.2 Hz, 1H), 4.08 - 4.14 (m, 2H), 4.61 (dd, J =7.2,12.4 Hz,1H), 4.81 (t, J=4.7 Hz, 1H), 5.14 (d, J=4.2 Hz, 1H), 5.31 (d, J=6.9 Hz, 1H), 8.81 (s, 1H) ), 6.06 (d, J = 4.4 Hz, 1H), 6.21 (d, J = 7.7 Hz, 1H), 6.74 (d, J = 3.7 Hz, 1H), 7.35 - 7.43 (m, 4H), 7.88 (d) , J = 3.7 Hz, 1H), 9.04 (s, 1H).

Example 26 (2R,3S,4R,5R)-2-[(R)-(4-chlorophenyl)-hydroxy-methyl]-5-(4-methylpyrrolo[2,3-d]pyrimidin-7 -yl)tetrahydrofuran-3,4-diol

Stirring (R)-[(3aR,4R,6R,6aR)-2,2-dimethyl-4-(4-methylpyrrolo[2,3-d]pyrimidin-7-yl) at room temperature -3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]-(4-chlorophenyl)methanol (0.500 g, 1.08 mmol) , a solution of 4 N HCl in 1,4-dioxane (50 mL) and MeOH (50 mL). After 1 hour at room temperature, the solution was concentrated under a stream of N ₂ to remove solvent. DCM (20 mL) and water (20 mL) were added to the residue. Extract with DCM and discard. The pH of the aqueous phase was adjusted to pH ~ 10 with a saturated aqueous solution of bicarbonate and extracted with DCM (4×20 mL). The organic extracts were combined, dried over sodium sulfate, filtered and evaporated. The residue was taken up in EtOAc (EtOAc)EtOAc. The filtrate was removed under reduced pressure to give crystall ^{ES / MS m / z (35} Cl / 37 Cl) 376.2 / 378.2 [M + H] +. _{^{1 H NMR (400MHz, d 6}} -DMSO) δ 2.63 (s, 3H), 3.95-3.97 (m, 1H), 4.05-4.08 (m, 1H), 4.53-4.59 (m, 1H), 4.75-4.79 ( m, 1H), 5.09 (d, 1H, J = 4.4 Hz), 5.25 (d, 1H, J = 7.0 Hz), 6.07-6.13 (m, 2H), 6.75 (d, 1H, J = 3.6 Hz), 7.31-7.34 (m, 2H), 7.36-7.40 (m, 2H), 7.76 (s, 1H, J = 3.6 Hz), 8.62 (s, 1H).

The results of the analysis below show that the compounds exemplified herein are useful as PRMT5 inhibitors and are useful in the treatment of cancer.

PRMT5/MEP50 SPA Analysis

The purpose of this analysis is a measure of inhibition by a test compound PRMT5 / MEP50 catalytically active complex in vitro to the ability of inhibiting the activity of PRMT5, this radiolabeled by the methyl - transferred to a derived from human histone H4 ³ H The receptor peptide of the N-terminal sequence is indicated.

The PRMT5/MEP50 enzyme complex was expressed using a baculovirus/Sf9 system and purified using anti-FLAG affinity chromatography (substantially as described in the preparation below, Stephen Antonysamy, Zahid Bonday, Robert M. Campbell, Brandon Doyle, Zhanna Druzina, Tarun Gheyi, Bomie Han, Louis N. Jungheim, Yuewei Qian, Charles Rauch, Marijane Russell, J. Michael Sauder, Stephen R. Wasserman, Kenneth Weichert, Francis S. Willard, Aiping Zhang and Spencer Emtage; Crystal structure of The human PRMT 5: MEP50 complex, Proceedings of the National Academy of Sciences, 2012, Vol. 109, pp. 17960-17965). The purified enzyme was diluted to 6.67 nM working stock in assay buffer (50 mM TRIZMA ^® Preset pH 8.5, 1 mM DTT, 0.005% TWEEN ^® 800.01% HSA).

The enzyme (15 [mu] l / well) was added to the 384 well assay culture plates (CORNING ^® catalog number 3706) in. Test compounds from a 10 mM DMSO stock solution were added to the culture plates in serial dilutions in assay buffer to achieve a final test concentration ranging from 50.0 [mu]M to 25 nM. A reaction mixture was established in assay buffer containing 1 μM histone H4 biotin-labeled peptide substrate prepared by ABC 431 peptide by FastMoc chemistry, H-SGRGKGGKGLGKGGAKRHRKVLRDK-Biotin (SEQ ID NO: 1), and 4 μM ³ H-SAM (15Ci / mmol, 0.366mCi / mL, 37μM, PERKIN-ELMER ® catalog number NET155000MC). 5 μL of peptide/ ³ H-SAM reaction mixture was added to each well of the culture plate to obtain the following final analysis conditions: 5 nM enzyme, 1 μM ³ H-SAM, 250 nM peptide, and 50 μM maximum test compound concentration with 0.5% DMSO at 20 μL final Under the volume. The analyte was incubated for 2 hours at room temperature. The reaction was stopped by the addition of HCl (5 M, 20 μL, Sigma Cat. No. G3272).

At 1mg / mL streptavidin-biotin YSI SPA scintillation beads (PERKIN-ELMER ^®, No. RPNQ0012) suspended in pH 8.5 containing 5 M guanidine HCl of the Tris buffer. 20 μL of this bead suspension was added to each well, and the resulting mixture was stirred and incubated at room temperature for 1 hour, after which the radioactivity was measured using a microplate liquid scintillation counter as an indicator for forming a methylated peptide product.

Raw data is generated from a radiometric measure such as count value per minute (CPM). An uninhibited control (DMSO) and a maximum inhibition control (250 [mu]M dehydrosinefungin, (substantially as Berry, DR & Abbott, BJ Incorporation of carbon-14-labeled compounds into sinefungin (A9145), a nucleoside antifungal antibiotic. J. Antibiot. 1978 (31) 185-191 and the references cited therein for isolation)). Percent inhibition was calculated as follows for each test compound concentration relative to control: % inhibition = [intermediate CPM (uninhibited control) - intermediate CPM (test compound)] / [intermediate CPM (uninhibited control) - intermediate CPM (control of maximum inhibition)]*100

Data were plotted in % inhibition (y-axis) compared to the log test compound concentration (x-axis) using ActivityBase XE or Genedata software. Using a 4-parameter curve fitting algorithm ₅₀ calculates the value of IC.

This analysis showed that all of the illustrated compounds inhibit PRMT5 / MEP50 catalytic activity, wherein the IC ₅₀ of less than 500nM. Additionally, Examples 1-3 were tested essentially as described above and exhibited the following activity for PRMT5/MEP50 as shown in Table 1.

Mean ± SEM; SEM = standard error of the mean

Agreement for 7 days of proliferation

The purpose of this analysis was to measure the ability of test compounds to inhibit cancer cell proliferation.

On day 1, A375 cells (ATCC, DMEM high glucose/10% Hi FBS) were cultured at 250 cells/well (100 μL/well) in a 96-well assay plate (BD Falcon 35-3219). The plates were incubated for 18-24 hours in a 37 ° C incubator (5% CO ₂ ). On day 2, test compounds were prepared in a medium from compound stock solutions (10 mM in 100% DMSO) and serially diluted (1:2 10 point serial dilution). 10 μL of serially diluted test compound was added to the cell culture dish (0.2% DMSO final concentration). 10 μL of the reference compound (Example 1) was added to row 1 of the cell culture dish (final 20 μM, 0.2% DMSO). The cell culture dishes were incubated for 7 days at 37 ° C / 5% CO ₂ . On day 9, melt the CELL TITER-GLO ^® (Promega No. G7571) buffer and equilibrate to room temperature before use. Similarly, prior to use lyophilization CELL TITER-GLO ^® by mass equilibrate to room temperature. Transferring the CELL TITER-GLO ^® buffer to amber bottle receiving quality, lyophilization so that the enzyme / substance mixture by restoration to form reagent CELL TITER-GLO ^®. The cell culture plates were equilibrated to room temperature for 5-10 minutes. The medium in the cell culture tray was gently removed by tapping and pipetting on a standard paper towel. Added to each well reagent CELL TITER-GLO ^® (25μL). The plates were incubated for 10-20 minutes at room temperature and then read. Luminescence was measured using a PERKIN-ELMER ^® ENVISION ^® multi-label plate reader. By 4-parameter curve fit analysis using the light-emitting active substrate materials, to give ₅₀ values of cell proliferation IC.

The results of this analysis show that Examples 1-3 have anti-proliferative effects on tumor cells based on their ability to inhibit PRMT5 activity. Proliferation IC ₅₀ values obtained are shown in Table 2:

Mean ± SEM; SEM = standard error of the mean

5/6 qPCR analysis of MDM4 exon in A375 tumor cells

The purpose of this analysis was to measure the PRMT5 dependence of MDM4 in A375 melanoma cells by measuring the test compound by measuring the ratio of exon 5 and 6 to MDM4 mRNA with exon 5 only. The ability of alternative splicing to demonstrate the ability of test compounds to inhibit PRMT5 function in cancer cells.

Having a growth medium (DMEM; HYCLONE ^TM with 10% FBS number of SH30022; GIBCO ^® 10082-147 or equivalent) of T150 flask, the A375 tumor cells (ATCC) - 70% of melanoma cancer cell lines were cultured to -90% confluence. Cells were treated with standard trypsin/EDTA for 3 minutes and aspirated and washed with PBS to release adherent cells from the culture flask. The cells were seeded at 5,000 cells/well in growth medium (90 μL) in a 96-well culture dish (Costar 3596). At 37 [deg.] C and 5% CO ₂ culture plate incubated overnight, and the final serially diluted (20,6.67,2.22,0.74,0.247,0.082,0.027,0.009,0.003,0.010μM, 0.2% yield of the test compound The cells were treated with DMSO for 72 hours. Activity was measured relative to maximal (2.0 [mu]M Example 2) and minimal (0.2% DMSO) controls.

On day 5 after the incubation, the medium was removed from the culture plate and the cells were washed twice with cold PBS (150 μl/well). DNA was diluted to 1/100 by the enzyme into a lysis solution, since the TAQMAN ^® CT gene expression, cell-to-kit (INVITROGEN ^TM Cat # AM1729) lysis working reagent is prepared. The lysis working reagent (50 μl/well) was added to the cell culture dish, the wells were mixed, and incubated for 5 minutes at room temperature. A set of stop solution (5 [mu]L) was added to each well, the wells were mixed and incubated for 2 minutes at room temperature. The reverse transcriptase parent mixture was prepared in the following volume ratio; 62.5: 31.25: 6.25 RT buffer: nuclease free water: RT enzyme. The RT mixture was added (48 microliters / well) to each well of a 96-well culture plate NUNC ^TM (THERMO SCIENTIFIC ^TM No. 260 860) of the. The RT mixture (20 μL) and each cell lysate sample (5 μL) were added to the 384 PCR culture plate (optical transparent reaction plate, catalog number 4309849, APPLIED BIOSYSTEMS ^® ) quadrant 1 and the RT mixture was combined with the cell lysate. Two additives were added to quadrant 3. For the RT reaction, the plates were sealed and then placed in a thermocycler set at 37 °C for 60 minutes; 95 °C for 5 minutes; and terminated at 4 °C. For qPCR, MDM4 exon 5 primer (Life Technologies Hs00967240-m1) and exon 6 primer (Life Technologies Hs00967242) were prepared at a volume ratio of 10:6:1 RT mixture:H ₂ O:exon probe, respectively. -m1). Exon 5 (17 μL) was added to odd rows of 384 PCR plates and exon 6 (17 μL) was added to even rows. cDNA (3 μL from the RT plate) was added to each well of a qPCR plate containing 17 μL of the exon primer solution by quadrant stamping. In this way, qPCR was performed on cDNA from a single RT reaction on exons of individual cell lysates. The plates were sealed, spun, and placed on an instant PCR instrument (Life Technologies ViiA7 Real Time PCR). In the following operating cycle TAQMAN ^® reaction stages: Stage 1 (50 ℃, 2 minutes), phase 2 (95 ℃, 10 minutes), Stage 3 (95 ℃, 15 seconds), Stage 4 (60 ℃, 60 sec) , in which stages 3 and 4 are repeated for 40 cycles.

The CT data was taken from the real-time PCR reader and the following calculations were performed using the Excel template, where "CPD" is the value of the sample treated with the compound and "DMSO" is the smallest control sample: (Exon 6 CT CPD - Explicit Sub 6 CT DMSO)-(exon 5 CT CPD-exon 5 CT DMSO)=△CT

2 ^(-△CT) = fold change

Software was plotted using Genedata fold change (y-axis) as compared to Log test compound concentration (x-axis) in the form of data, using the four parameter logistic fit _IC50 values _are calculated EC.

This analysis indicated that all of the exemplified compounds tested in this assay inhibited the splicing of MDM4 by PRMT5 as indicated by the ratio of mRNA prepared with exon 6 compared to mRNA with exons 5 and 6. Guide regulation.

For example, the resulting capacity EC for Example 1, 2 and 3 of the adjusting splicing MDM4 ₅₀ values shown in Table 3.

Mean ± SEM; SEM = standard error of the mean

Combination with BCL2 inhibitor ABT-199 in AML cell lines (cell proliferation analysis on days 4 and 7)

The purpose of this analysis was to demonstrate synergistic inhibition of proliferation of AML cell lines when PRMT5 inhibitors were combined with the BCL2 inhibitor ABT-199 (Abbot Laboratories).

On day 1, GDM-1 [ATCC ^{® was} maintained at 4000 and 10000 cells/well (100 μL/well) in 96-well assay plates (BD FALCON ^® 35-3219), maintained throughout GDM-1 In the assay medium: RPMI-1640 medium/20% heat-inactivated FBS (GIBCO ^® ) and EOL-1 cells [ATCC ^® , maintained throughout the EOL-1 assay medium: RPMI-1640 medium/10% heating is not activated FBS (GIBCO ^® )]. The plates were incubated for 18-24 hours in a 37 ° C incubator (5% CO ₂ ). On day 2, 2.0% DMSO was prepared using assay medium (for GDM-1 and EOL-1, as indicated above or RPMI-1640/20% heat-inactivated FBS and RPMI-1640/10% FBS, respectively). And 100 μL was added to rows 3-12 in the preparation plate. 10 μM of test compound ABT-199 in 2% DMSO, and 25 μM of PRMT5 inhibitor Example 1 in 2% DMSO were prepared and dispensed in wells A2 and B2, respectively, followed by serial dilutions (1:3, 10, respectively). Point) to holes A11 and B11. For combination treatment, in wells C2-C11 (0.1 μM) to H2-H11, in assay medium (for GDM-1 and EOL-1, FBS as indicated above or RPMI-1640/20% heat inactive, respectively) In the RPMI-1640/10% FBS), test compound examples were serially diluted 1:2, and then test compound ABT-199 was serially diluted 1:3 from C2-H2 (1.0 μM) to C11-H11. 20 μM staurosporine (available in Sigma) in 2% DMSO was prepared as a reference compound in wells A1 to H1. 11 μL of the serially diluted compound was added to a well containing 100 μl of cells in a cell culture dish. The final starting concentration in the cell culture dish was as follows: for a single compound, Example 1 of the PRMT5 inhibitor was 2.5 μM and ABT-199 was 1 μM; for the combination of the two compounds, the initial concentration of the PRMT5 inhibitor Example 1 was 0.01 μM and ABT-199 was 0.1 μM; the final DMSO concentration was 0.2%. The cell culture dishes were incubated for four or seven days at 37 ° C / 5% CO ₂ . On day 6 or day 9 (for the fourth or seventh day of the test, respectively), CELL TITER-GLO ^® (Promega No. G7571) buffer was thawed and equilibrated to room temperature prior to use. Similarly, prior to use lyophilization CELL TITER-GLO ^® by mass equilibrate to room temperature. Transferring the CELL TITER-GLO ^® buffer to amber bottle receiving quality, lyophilization so that the enzyme / substance mixture by restoration to form reagent CELL TITER-GLO ^®. The cell culture plates were equilibrated to room temperature for 5-10 minutes. CELL TITER-GLO ^® Reagent (100 μL) was added to each well. The plates were incubated for 10-15 minutes at room temperature and then read. Luminescence was measured using a PERKIN-ELMER ^® ENVISION ^® multi-label plate reader. Flux analysis data back with a medium, to obtain IC ₅₀ values and based on proliferation inhibition of Chou-Talalay combination index analysis. [Chou TC, Talalay P. Analysis of combined drug effects: a new look at a very old problem. Trends Pharmacol Sci 1983; 4: 450-4.]

The results of this analysis indicate that the combination of the BCL2 inhibitor ABT-199 and the PRMT5 inhibitor Example 1 has a synergistic inhibitory effect on cancer cell proliferation. The resulting IC ₅₀ values and the absolute proliferation Combination Index values shown in Table 4:

Mean ± standard error of the mean

Analysis of 5/6 RNA splicing of MDM4 exon in mouse A375 xenograft tumor

The purpose of this analysis was to quantify the ability of test compounds to inhibit PRMT5-mediated stagnation of exon 6 of treated MDM4 mRNA in A375 tumor cells in a mouse tumor xenograft model. This analysis presents the effect of test compounds on specific PRMT5 mediated splicing events in tumors in animal models.

In the incubator (5% CO ₂₎ at 37 ℃, the growth of A375 cells in medium (DMEM high glucose / 10% Hi FBS) manipulation 18-24 hours. Cells were mixed with MATRIGEL ^® (1:1) and cells (5×10 ⁶ /animal) were subcutaneously implanted into the back of the ventral flank of mice (female nude mice, Harlan). The implanted tumor cells grow into solid tumors. Tumor volume and body weight were measured twice a week using a caliper. After the tumor volume reached approximately 200-250 mm ³ (approximately 20 days after implantation), the animals were randomized and divided into compound treatment groups. Test compounds were administered by oral gavage (disposed in SWFI with 1% HEC/0.25% TWEEN ^® 80/0.05% antifoam). The test compound dose is in the range of 3 to 100 mg/kg. Mice were sacrificed 4 hours after the last dose (4 days of dosing).

Tumor tissue was harvested and homogenized as described below. Tumor samples were placed in a bowl and add 1mL liquid N _2. Squeeze the tumor in a bowl. Small fragments of tumor (about 20 mg each) were transferred to 2 lysis matrix D tubes (MPBIO Cat. No. 6913-250). One tube was used for RNA processing and the other was used for protein lysate preparation. For RNA processing, using the Bio101 FastPrep FP120 homogenizer (setting 6) in the micro RNEASY ^® kit (QIAGEN-74104) extraction buffer (0.5 mL each) of tumor tissue manipulation homogenized for 30 seconds. Total RNA was purified using QUARTZY ^® RNEASY ^® Micro Suite-50 (QIAGEN-74104, RNEASY ^® Micro Manual, Fourth Edition, June 2012). Check RNA concentration to ensure OD 260/280nM 1.9. For cDNA synthesis, A&B APPLIED BIOSYSTEMS ^® , a high capacity cDNA reverse transcription kit (Catalog No. 4368813) was used. In 20μL containing 10 × RT buffer -2.0μL; 25 × dNTP mixture (100mM) -0.8μL; 10 × RT random primers -2.0μL; MULTISCRIBE ^TM reverse transcriptase -1.0μL; nuclease-free H ₂ O-4.2 of 1 μg of total RNA (10 μL) was used in each RT reaction volume of μL. For use with the high capacity cDNA reverse transcription kit, these conditions (from the APPLIED BIOSYSTEMS ^® thermal cycler) are optimal: Step 1 - Temperature 25 ° C / time 10 minutes; Step 2 - Temperature 37 ° C / time 120 minutes ; Step 3 - Temperature 85 ° C / time 5 minutes; Step 4 Temperature 4 ° C until further use. Subsequently, 20μL containing 10μL 2 × PCR mixture, [mu] L probe, cDNA was prepared via 3 L (20ng) and water (6 pL) of the total volume, operating TAQMAN ^® qPCR reaction in thermal cycler program. [Absolute QPCR ROX mixture from blue THERMO SCIENTIFIC ^TM of (2 ×), Cat #: AB-4139; MDM4 from APPLIED BIOSYSTEMS ^® of (exon 5 and exon 6) probes, respectively, catalog number: Hs00967240 -m1 and catalog number: Hs00967242-m1; GAPDH probes, catalog number from APPLIED BIOSYSTEMS ^® of: Hs02758991-g1; CDKN1A of from APPLIED BIOSYSTEMS ^® (of P21) probes, catalog number: Hs02758991-g1]. Sample operation on ViiA7 TAQMAN ^® thermocycler machine (APPLIED BIOSYSTEMS ^®).

Western blot analysis of mouse A375 tumor xenograft MDM4 and SmD1-me2S

For Western blotting, homogenize the tumor tissue in 500 μL of a set of XY lysis buffer including a complete small protease inhibitor cocktail (Roche, catalog number: 11 836 170 001) [This mixture contains 10 mg /mL anti-plasmin peptide hemisulfate (Sigma catalog number L2884); 10 mg/mL trypsin-chymotrypsin inhibitor (Sigma catalog number T9777); 10 μg/mL TPCK (Sigma catalog number T4376); 10 μg/ mL aprotinin (Sigma Catalog No. A1153); 60mM β-glycerophosphate disodium salt dihydrate (Sigma catalog number ^{G9891); 1% TRITON TM X} -100 (Sigma Catalog No. T9284); 25mM Tris pH ~ 7.5 (INVITROGEN TM directory No. 15567-027); 2.5 mM disodium diphosphate (Fluka Cat. No. 71501); 300 mM NaCl ₂ (Mallinckrodt Cat. No. 7581); 2 mM TAME (Sigma Cat. No. T4626); 15 mM pNPP (pNPP disodium salt hexahydrate, Sigma Cat. No. P4744); 5 mM benzamidine hydrochloride hydrate (Sigma catalog number B6506); 10 mM sodium fluoride (Sigma catalog number S-7920); 1 mM anhydrous sodium metavanadate (Sigma catalog number 59098); 5 g 1 mM DTT ( Sigma Cat. No. D9779); 15mM EDTA pH ~ 8.0 (INVITROGEN TM Catalog No. 15575020); 5mM EGTA pH ~ 8.0 (Sigma mesh No. E3889); 1mM microcystin LR (Fisher / Axxora Catalog No. A350012m001); 0.25mM Pefa Bloc (Sigma Cat. No. 76307)]. The protein lysate was spun down at 8,000 rpm for 10 minutes and the supernatant layer was transferred to a 1.5 mL tube for sonication (amplitude 25% yield) for 15 seconds. The lysate was again rotated down at 15,000 rpm for 10 minutes. 60 μg of each protein lysate was obtained from the supernatant layer, and SDS-PAGE sample loading buffer [15 μL protein lysate; 5 μL 4× sample buffer (Bio-Rad catalog number 161-0791); 2.25 μL 10× sample reduction Agent (INVITROGEN ^TM Cat. No. NP0004); boil the sample at 100 ° C for 10 minutes] mixed, and operated in SDS-PAGE using 4-20% Tris-glycine gel - 1.5 mm × 15 well (INVITROGEN ^TM ) . Once the dye front reaches the bottom of the gel, the electrophoresis is terminated and the protein is transferred from the gel to the nitrocellulose membrane for Western blotting with the following primary antibody and with rabbit anti-GAPDH antibody (catalog: AB9485 Abcam): anti-MDM4 pure line 8c6, a mouse strain from Millipore, catalog number 04-1555; anti-SmD1-me2S [mouse individual, 2.14 mg/mL]. Anti-SmD1-me2S monoclonal antibodies were generated in ENZ Lilly in NZB/W mice (The Jackson Laboratory). Five 6-week-old female mice were immunized subcutaneously with 75 μg of a mixture of two peptides KLH bound to complete Freund's adjuvant (see below).

CREAVAGR*GR*GR*GR*GR*GGPRR(SEQ ID NO:2)

REAVAGR*GR*GR*GR*GR*GGPRRC(SEQ ID NO:3)

The asterisk indicates a symmetric dimethyl-Arg. KLH binds to Cys amino acid (C). After initiation, mice were injected subcutaneously with a mixture of 50 [mu]g of two peptides bound to KLH in incomplete Freund's adjuvant every 3 weeks. Ten days after the third booster immunization, blood was collected using a post-mortem procedure to measure the antibody titer in the serum. Spleen cells from positive mice were isolated and fused with myeloma cell line P3X63Ag8.653 (ATCC ^® CRL-1580) using PEG at a ratio (1:4), as by Harlow (Antibodies: a laboratory manual. Cold Spring Harbor, NY) , 2004) described. The fused cells were seeded in a 96-well culture dish and tested for binding to the SmD1-me2S peptide by ELISA one week after selection in the selection medium containing the HAT and serum layers. Specificity was tested by ELISA with unmethylated peptide and SmD3-symmetric dimethyl-Arg peptide. The ELISA-positive fusion tumor cells were subcultured by serial dilution, and the antibody was purified by affinity chromatography using the above peptide antigen.

Once the transfer is complete, block the membrane with blocking buffer (Odyssey blocking buffer with 0.05% TWEEN ^® -20, Biorad Cat. No. 161-0781, LI-COR Biosciences catalog number 927-40000), and then at 4 °C Anti-MDM4 pure line 8c6 (single mouse from Millipore, catalog number 04-1555) diluted 1:500 in blocking buffer, anti-SmD1-me2S diluted 1:300 (self-produced mice) Single plants, 2.14 mg/mL), and treated with a 1:1000 dilution of rabbit anti-GAPDH antibody (catalog: AB9485 Abcam) to treat the membrane overnight. TBS TWEEN ^® membrane was washed 3 times (0.05% yield), 10-15 minutes each [10 × TBS (Sigma Catalog No. 170-6435)]. The membrane was then incubated with the following secondary antibody at room temperature for 1.5 hours in the following buffer: IRDye 680LT, goat anti-rabbit, LICOR ^® catalog number 926-68021, lot number C10314-03, press 1:6000 diluted, and IRDye 800CW, goat anti-mouse IgG (H + L), LICOR ® Cat. No. 926-32210, lot C10131-01, diluted 1: 6000. The membrane was washed 3 x (10 minutes each) with TBS TWEEN ^® . The film is developed with LICOR ^® ODYSSEY ^® imaging system for scanning to measure the amount of Western blot detection of the article with strength. Percent inhibition was calculated for the group treated with the test compound relative to the vehicle treated group sample as the minimum inhibition group using the following equation: % inhibition = [intermediate band strength (control without suppression) - intermediate band strength (test) Compound)]/[Intermediate Band Strength (Control Uninhibited) - Intermediate Band Strength (Maximum Inhibition Control)]*100

The maximal inhibition control sample was selected from A375 tumors, which were obtained in vitro from tumor-bearing mice 4 days after oral administration of Example 10 10 mg/kg BID.

The ED ₅₀ calculated for the study at this time to reach 50% of the dose from the dose response effect required.

For the described analysis (mouse A375 xenograft tumor MDM4 exon 5/6 RNA splicing analysis and mouse A375 tumor xenograft MDM4 and SmD1-me2S western blot analysis), the compounds of Examples 1, 2 and 3 were at dose 30 mg/kg PO QD or BID gives up to 50% effect. These results indicate that the exemplified compounds of Examples 1, 2 and 3 inhibit PRMT5 activity in vivo, as indicated by their effect on the PRMT5 receptor and the associated molecular biology endpoints.

Xenograft tumor model

The purpose of this analysis was to measure the reduction in tumor volume in response to administration of the test compound in a mouse cancer model.

Cell growth (melanoma) in DMEM with 10% HI FBS (GIBCO ^® Cat. No. 10082-147) the / high glucose (HYCLONE ^TM Cat. No. SH30022) manipulation A375. Will-2 (DLBCL) cells were grown in RPMI 1640 with L-glutamic acid and 20% heat-inactivated FBS. Namalwa (Burkitt's Lymphoma) cells (ATCC) in RPMI 1640 medium supplemented with L-glutamic acid, 25 mM HEPES (GIBCO ^® 22400-089), 1 mM sodium pyruvate and 7.5% FBS Growing. Molt 4 cells were grown in RPMI 1640 with L-glutamic acid, 25 mM HEPES [GIBCO 22400-089], 1 mM sodium pyruvate and 10% FBS. Cells were harvested and injected subcutaneously into the posterior flank of nude mice (A375: 5 x 10 ⁶ cells/animal, mixed with MATRIGEL ^® 1:1; Will-2; 1 x 10 ⁷ cells/animal; Namalwa: 2 x 10 ⁶ cells/animal; Molt 4: 5 x 10 ⁶ cells/animal, mixed with MATRIGEL ^® 1:1). When tumors were established (7-21 days after implantation, approximately 200 mm ³ ), animals were randomized and divided into control and test groups. Test compounds were formulated in 1% HEC/0.25% TWEEN ^® -80/0.05% antifoam. Test compounds and vehicles are administered by oral gavage. Tumor response was determined by tumor volume measurements (gauge gauge) performed twice a week during the course of the treatment and reported as percent inhibition of tumor volume compared to the vehicle control group.

Example 1 demonstrates dose-dependent anti-tumor activity in all three xenograft tumor models. For example, in the melanoma model (A375), 64% inhibition was achieved when administered on day 4 and discontinuation schedule on day 3 was administered at 10 mg/kg QD for 26 days; when in the same row When administered at 15 mg/kg, 71% inhibition was achieved. In the DLBCL tumor model (WILL-2), 50% degradation was achieved when administered at 2 mg/kg (BID for 14 days); 74% inhibition was achieved when administered at 5 mg/kg (QD for 14 days) . In the lymphoma tumor model (Namalwa), when administered at 2 mg/kg (BID for 14 days), 74% inhibition was achieved; When administered at mg/kg (QD for 14 days), 61% inhibition was achieved. This data indicates that Example 1 inhibited tumor xenograft growth in the above three tumor models.

In the acute lymphoblastic leukemia model (Molt4), Example 4 achieved a -75% inhibition when administered at 60 mg/kg (BID for 30 days). This data indicates that Example 4 inhibits tumor xenograft growth in the above tumor models.

Preferably, the compounds of the invention are formulated as pharmaceutical compositions for administration by a variety of routes. Most preferably, the compositions are for oral or intravenous administration. Such pharmaceutical compositions and methods of making same are well known in the art. See, for example, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (edited by D. Troy et al., 21st ed., Lippincott Williams & Wilkins, 2005).

As used herein, the term "effective amount" refers to a compound of the invention or a pharmaceutically acceptable salt thereof that provides a desired effect in a patient under diagnosis or treatment when a single or multiple doses are administered to a patient. The amount or dose.

The effective amount can be readily determined by the attending diagnostician (as known to those skilled in the art) by using known techniques and by observing the results obtained under similar circumstances. In determining an effective amount for a patient, the attending diagnostician considers a number of factors including, but not limited to, the type of mammal; its size, age, and overall health; the particular disease or condition involved; The severity of the disease or condition; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the formulation being administered; the dosage regimen selected; the use of the concomitant medication; and other relevant circumstances.

The compounds of the invention are generally effective over a wide dosage range. For example, the dose/day is normally in the range of about 0.05-1000 mg per day. Preferably, the doses are in the range of from 0.1 to 500 mg per day. More preferably, the doses are in the range of from 0.5 to 100 mg per day. In some cases, doses below the lower limit of the foregoing range may be excessive, while in other cases, still larger doses may be employed without causing any deleterious side effects, and thus the above dosage ranges are not intended to be limiting in any way. The scope of the invention. Will understand, actually vote The amount of the compound will be determined by the physician, in view of the circumstances, including the condition to be treated, the route of administration selected, the actual compound administered, age, weight, and the response of the individual patient and the severity of the patient's symptoms.

<110> American Lilly Pharmaceuticals

<120> 5'-substituted nucleoside compound

<130> X20426

<140> 15382225.9

<141> 2015-05-04

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 26

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<220>

<221> MOD_RES

<222> (26)..(26)

<223> Location 26 of the amino acid labeled with biotin

<400> 1

<210> 2

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<220>

<221> MOD_RES

<222> (8)..(8)

<223> Arg at position 8 is symmetric dimethyl-Arg

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Arg at position 10 is symmetric dimethyl-Arg

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Arg at position 12 is symmetric dimethyl-Arg

<220>

<221> MOD_RES

<222> (14)..(14)

<223> Arg at position 14 is symmetric dimethyl-Arg

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Arg at position 16 is symmetric dimethyl-Arg

<400> 2

<210> 3

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<220>

<221> MOD_RES

<222> (7)..(7)

<223> Arg at position 7 is symmetric dimethyl-Arg

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Arg at position 9 is symmetrical dimethyl-Arge

<220>

<221> MOD_RES

<222> (11)..(11)

<223> Arg at position 11 is symmetrical dimethyl-Arg

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Arg at position 13 is symmetric dimethyl-Arg

<220>

<221> MOD_RES

<222> (15)..(15)

<223> Arg at position 15 is symmetrical dimethyl-Arg

<400> 3

Claims (17)

a compound having the formula: Wherein: R ^1a is hydrogen, C ₁ -C ₂ alkyl, C ₃ -C ₆ cycloalkoxy, trifluoromethyl, cyano, chloro or fluoro; R ^1b is hydrogen, chloro or fluoro; R ² is hydrogen Or C ₁ -C ₃ alkyl; and R ³ is hydrogen, methyl or amine; or a pharmaceutically acceptable salt thereof. The compound of claim 1 or a salt thereof, which has the formula: Wherein: R ^1a is hydrogen, C ₁ -C ₂ alkyl, C ₃ -C ₆ cycloalkoxy, trifluoromethyl, cyano, chloro or fluoro; R ^1b is hydrogen, chloro or fluoro; R ² is hydrogen Or C ₁ -C ₃ alkyl; and R ³ is hydrogen, methyl or amine; or a pharmaceutically acceptable salt thereof. The compound of claim 1 or 2, or a salt thereof, wherein the configuration of the palmitic carbon to which the R ² substituent is attached is R, S or a mixture thereof. The compound of claim 1 or 2, or a salt thereof, which is Or a pharmaceutically acceptable salt thereof. The compound of claim 4, or a salt thereof, which is crystalline and characterized by an X-ray powder diffraction pattern (Cu radiation, λ = 1.54060 Å), the X-ray powder diffraction pattern comprising a peak of 25.1°, and one or more The peaks are selected from the group consisting of 17.0°, 13.6°, 20.5°, 24.0°, and 14.5° (2θ±0.2°). The compound of claim 1 or 2, or a salt thereof, which is Or a pharmaceutically acceptable salt thereof. The compound of claim 1 or 2, or a salt thereof, which is Or a pharmaceutically acceptable salt thereof. The compound of claim 1 or 2, or a salt thereof, which is Or a pharmaceutically acceptable salt thereof. The compound of claim 1 or 2, wherein R ^1a is hydrogen, chloro or cyclopropoxy, or a salt thereof. The compound of claim 1 or 2, wherein R ^1b is hydrogen or chlorine, or a salt thereof. The compound of claim 1 or 2, wherein R ² is hydrogen or methyl, or a salt thereof. The compound of claim 1 or 2, wherein R ³ is an amine group, or a salt thereof. A compound of claim 1 or 2, or a salt thereof, for use in therapy. A compound according to claim 1 or 2, or a salt thereof, for use in the treatment of cancer. The compound of claim 14 or a salt thereof, wherein the cancer is selected from the group consisting of glioblastoma, melanoma, sarcoma, gastric cancer, pancreatic cancer, cholangiocarcinoma, bladder cancer, breast cancer, non-small cells Lung cancer, leukemia and lymphoma including acute myeloid leukemia. A pharmaceutical composition comprising a compound according to any one of claims 1 to 12, or a salt thereof, and a pharmaceutically acceptable excipient, carrier or diluent. Use of a compound according to any one of claims 1 to 12, or a salt thereof, for the preparation of a medicament for the treatment of cancer, wherein the cancer is selected from the group consisting of glioblastoma, melanoma , sarcoma, gastric cancer, pancreatic cancer, cholangiocarcinoma, bladder cancer, breast cancer, non-small cell lung cancer, leukemia including acute myeloid leukemia and lymphoma.